Glial cell line-derived neurotrophic factor receptors

ABSTRACT

Receptors for Glial Cell Line-Derived Neurotrophic Factor (GDNF), their cellular expression, isolation, biochemical characterization, and sequences are disclosed. c-RET is disclosed as one receptor for GDNF; additional novel receptors are also disclosed. The preparation of monoclonal antibodies directed against GDNF is also disclosed.

[0001] The present application is a continuation-in-part of applicationSer. No. 08/747,842, pending, which claims priority benefit ofprovisional application serial Nos. 60/006,619, filed Nov. 13, 1995;60/015,767, filed Apr. 16, 1996; 60/021,965, filed Jun. 27, 1996;60/020,638, filed Jun. 27, 1996; and 20/020,639, filed Jun. 27, 1996,all applications hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to the identification of receptorsfor and functions of GDNF, and cell lines expressing the receptors.

BACKGROUND OF THE INVENTION

[0003] Glial cell line-derived neurotrophic factor (GDNF) is a trophicpolypeptide. It is a disulfide bridge-linked homodimer of two 134-aminoacids long glycosylated polypeptides, with a molecular weight ofapproximately 25-30 kD for each monomer. Prior to the molecular cloningof GDNF in 1993, investigators sought a trophic polypeptide which wouldalleviate the neuronal loss associated with Parkinson's disease,specifically dopaminergic neurons of the ventral mesencephalon. Thesurvival of this subpopulation of neurons has been known for some timeto be promoted by soluble factors present in the conditioned media ofglial cell lines. It was from one of these cell lines that the GDNFprotein was initially isolated based upon its ability to promotedopamine uptake in primary cultures prepared from embryonic ventralmidbrain neurons (Lin et al., 260 Science 1120, 1993). Subsequently,GDNF was shown to promote survival of adult substantia nigra neurons invivo following pharmacological treatments and lesions that mimicParkinsonian syndromes (Beck et al., 377 Nature 339, 1995; Tomac et al.,373 Nature 335, 1995) Although GDNF was originally reported to be highlyspecific for dopaminergic neurons, several other potent activities ofthis molecule have subsequently been demonstrated, including survivaland phenotypic responses in facial and spinal motor neurons (Hendersonet al., 266 Science 30 1062, 1994; Oppenheim et al., 373 Nature 344,1995; Yan et al., 373 Nature 341, 1995), noradrenergic neurons of thelocus coeruleus (Arenas et al., Neuron, in press, 1995), cerebellarPurkinjie cells (Mount et al., 92 PNAS 9092, 1995), sympathetic andsensory neurons in peripheral ganglia (Trupp et al., 130 J. Cell Biol.137, 1995) and for populations of peripheral neurons with target-derivedand paracrine mode of action (Trapp, M. et. al., J. Cell Biol., 130,137-148 (1995); Pitchel, J., Sariola, H., Hoffer, B. & Westphal, H.(unpublished observation); Buj-Bello, A., Buchman, V. L., Horton, A.,Rosenthal, A.& Davies, A. M. Neuron, 15, 821-828 (1995). As many ofthese neurons are affected in neurodegenerative diseases, GDNF may havepotent therapeutical applications. Particularly, exogenouslyadministered GDNF maintains dopaminergic neurons of the substantia nigrain experimentally induced Parkinsons disease in rodents (Beck et al.(1995) Nature, 373, 339-341; Tomac et al. (1995) Nature, 373, 335-339)and leads to functional recovery in Parkinsonian rhesus monkeys (Gash etal. (1996) Nature, 380, 252-255). GDNF treatment also rescues about halfof the experimentally axotomized murine motoneurons (Oppenheim et al.(1995) Nature, 373, 344-346; Li et al. (1995) Proc. Natl, Acad. Sci.U.S.A., 92, 9771-9775) suggesting that GDNF may be used in treatment ofmotoneuronal diseases. The studies of the mechanism of GDNF action innormal and pathogenic conditions have been, however, basically hamperedas its receptor was not known.

[0004] Based upon structural similarities (primarily seven conservedcysteine amino acid residues), GDNF appears to be a distant member ofthe transforming growth factor-beta (TGF-13) superfamily ofmultifunctional cytokines, which includes TGF-βs, activins,bone-morphogenetic proteins (BMPs) and growth and differentiationfactors (GDFS) (Roberts et al., 327 Philos.Trans.R.Soc.Land. 145,1990).TGF-β and related ligands are known to suppress proliferation inepithelial and immune cells, to function as morphogens in earlydevelopment, to induce ectopic expression of skeletal tissue, and topromote survival and differentiation of neurons. TGF-β superfamilyproteins interact with numerous receptor subunits on the surface ofresponsive cells (Attisano et al., 1222 Mol.Cell Res. 71, 1994; Derynck,19 Trends Biochem. Sci. 548, 1994). Different receptor types have beendescribed based on the molecular weights of affinity labeled Complexes.Among these are the type I, type II and type III receptors, whichrepresent binding proteins of 55 kD, 70 kD and 3 OOkD, respectively.Type III receptors are abundantly expressed transmembrane proteoglycansof approximately 3 OOkD with a short cytoplasmic tail, and are thoughtto function in recruitment of ligand to an oligomeric receptor complex(Lopez-Casillas et al., 67 Cell 785, 1991). Indeed, a type III receptoris required on some cell lines for TGF-β2 binding to the signalingreceptors. Type I and type II receptors are transmembrane proteins withan intracellular serine-threonine kinase domain and can thereforetransmit downstream signals upon ligand binding (Attisano et al., 75Cell 671, 1993; Derynck, 1994 supra). Type II receptors areconstitutively activated kinases which upon ligand binding recruit typeI receptors to a signaling complex. In this complex, type I receptorsare phosphorylated by type II receptors on a juxtamembrane domain richin serine residues, this phosphorylation is thought to result in theactivation of the ser-thr kinase activity of type I receptors and indownstream signaling (Wrana et al., 370 Nature 341, 1994). According tothis model, TGF-β superfamily proteins can not bind to type I receptorsin the absence of type II receptors, although in some cases, type Ireceptors are necessary for efficient binding to type II receptors(Letsou et al., 80 Cell 899, 1995). Multiple cDNA clones of type I, IIand III receptors for TGF-βS, activins and BMPs have been isolated byeither expression or homology cloning, including seven mammalian type Ireceptors, four type II receptors and one type III betaglycan receptor.Additional membrane proteins binding different members of this familyinclude glycosylphosphatidyl inositol (GPS)-linked 150 kD and 180 kDproteins of unknown structure and function (MacKay and Danielpour, 266J. Biol. Chem. 9907, 1991), and endoglin, a 180 kD disulphide linkeddimer which binds TGF-β1 but not TGF-β2.

[0005] The isolation and characterization of GDNF receptors is aprerequisite for the understanding of the full range of biologicalactions of GDNF and the signaling events that take place upon GDNFbinding to responsive cells. Until now, progress in this area has beenhampered by the lack of cell lines responsive to GDNF, that is, celllines comprising GDNF receptors.

SUMMARY OF THE INVENTION

[0006] Receptors for GDNF are disclosed herein, as are cell linesexpressing the same. Methods for identifying and isolating thesereceptors are also disclosed.

[0007] In one aspect, the present invention relates to isolatedreceptors which bind GDNF.

[0008] In another aspect, the present invention relates a method fordetermining compounds or compositions which bind GDNF receptors.

[0009] In yet another aspect, the present invention relates to methodsfor identifying homologs of GDNF by screening for compounds orcompositions which have similar biological effects, such as tyrosinephosphorylation, increase in c-fos mRNA, and increases in cell survival.

[0010] In still another aspect, the present invention relates to methodsfor identifying analogs of GDNF by screening for compounds orcompositions which are antagonistic for the biological effects of GDNF,such as are listed above.

[0011] In a further aspect, the present invention relates to compoundshaving the sequence as set forth in SEQ ID NOS:2 and 9.

[0012] In yet a further aspect, the present invention relates to nucleicacids having the sequence as set forth in SEQ ID NOS:5 and 10.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. Binding of ¹²⁵I-GDNF to receptors on chick sympatheticneurons. (a) Saturation steady-state binding of ¹²⁵I-GDNF to E10embryonic chick sympathetic neurons. Data are expressed as mean ±SD oftriplicate determinations. (b) Scatchard transformation of the dataplotted in (a). (c) Hill transformation of the data plotted in (a) nH:Hill coefficient.

[0014]FIG. 2. Affinity labeling of GDNF receptors on chick sympatheticneurons. ¹²⁵I-GDNF was cross-linked to E10 embryonic chick sympatheticneurons, receptor complexes were fractionated by SDS/PAGE and visualizedby gel autoradiography (middle lane). A doublet at 100 kD and a 300 kDcomplex are indicated by arrows. Excess cold GDNF preventedcross-linking of ¹²⁵I-GDNF (right lane). For comparison, crosslinking of¹²⁵I-TGF-β to mink lung epithelial cells MvILu is also shown (leftlane). Molecular weight markers are indicated in kD.

[0015]FIG. 3. Affinity labeling of GDNF receptors on cell lines.¹²⁵I-GDNF was cross-linked to C6 glioma, RN33B raphe nucleus, L6myoblast and MN-1 motor neuron cell lines with either DSS or EDAC ascrosslinker agents. Receptor complexes were fractionated by SDS/PAGE andvisualized by gel autoradiography. Excess cold GDNF preventedcross-linking of ¹²⁵I-GDNF (cold). Molecular weight markers areindicated in kD.

[0016]FIG. 4. Individual constituent affinities of GDNF receptorsubunits in RN33B and MN-1 cells. (a) Sizes of different GDNF receptorcomplexes on RN33B and MN-1 cells after cross linking with EDAC or DSS.(b) and (c) ¹²⁵I-GDNF was cross-linked to RN33B (b) or MN-1 (c) cells inthe presence of increasing concentrations of cold GDNF. The percentageof ¹²⁵I-GDNF binding to the indicated receptor subunit is plotted as afunction of the concentration of cold GDNF used during binding.

[0017]FIG. 5. Expression of GDNF mRNA in cell lines expressing GDNFreceptors. (a) Autoradiogram of an RNAse protection assay using equalamounts of total RNA from the indicated cell lines. Kidney post natalday 1 and yeast tRNA were used as positive and negative controls,respectively. (b) Quantification of the level of GDNF mRNA in differentcell lines relative to the level in PI kidney. undiff RN33B,undifferentiated RN33B cells; diff RN33B, differentiated RN33B cells;diff RN338+GDNF, RN33B cells differentiated in the presence of GDNF.

[0018]FIG. 6. Expression of mRNA for c-ret in different cell lines.

[0019]FIG. 7. GDNF stimulation of tyrosine phosphorylation of ERKs inRN33B and MN-1 cells. RN33B (a) or MN-1 (b) cell monolayers were exposedto 50 ng/ml GDNF during the indicated periods of time (in minutes), celllysates were fractionated by SDS/PAGE and Western blots probed with ananti-phosphotyrosine antibody (aP-Tyr). The blots were stripped andreprobed with an anti ERK2 antibody (a-ERK2) that recognizes bothp42^(erk) 12 and p44^(erk1) (arrows to the right). Molecular weightmarkers are indicated in kD.

[0020]FIG. 8. GDNF stimulation of c-fos mRNA expression in RN33B andMN-1 cells. RN33B (a) or MN-1 (b) cell monolayers were exposed to 50ng/ml GDNF during the indicated periods of times, total RNA wasextracted and fractionated in agarose gels and Northern blots probedwith a ³²P-labeled rat c-fos probe. Shown are x-ray autoradiograms offilters washed at high stringency.

[0021]FIG. 9. GDNF increased the survival of RN46A cells. RN46A cellswere differentiated in media ±0-50 ng/ml GDNF for 8 days. The datarepresent the means ±SEM of three independent experiments (1,500-3,000cells counted per condition). ANOVA indicated that the GDNF had asignificant effect on survival at all concentrations compared to mediaalone (overall ANOVA: df=6,203; F=1 1.39, p,0.001; unequal N LSD posthoc test, p.0.001).

[0022]FIGS. 10a-c. Biological and biochemical responses of MN-1 to GDNF.(a) GDNF stimulates survival of serum-deprived MN-1 cells. (B)GDNFstimulates rapid and transient tyrosine phosphorylation of severalproteins (asterisks) in MN-1 cells. Time of GDNF treatment (in minutes),and molecular weight markers are indicated. (c) Rapid and sustained ERK1and ERK2 tyrosine phosphorylation stimulated by GDNF in MN-1 cells.

[0023]FIGS. 11a-b. c-RET is a signal transducing receptor for GDNF. (a)Immunoprecipitation analysis of GDNF-receptor complexes in MN-1 cells.GDNF-labeled binding proteins could be precipitated with lectinSepharose beads, or antibodies against GDNF, phospho-tyrosine (P-Tyr)and c-RET. Control preimmune antibodies did not immunoprecipitate GDNFreceptor complexes. (b) GDNF induces tyrosine phosphorylation of c-RETin MN-1 cells. c-RET tyrosine phosphorylation was detected already 5minutes after addition of GDNF (upper panel). Saturation was observed at30 ng/ml GDNF (lower panel).

[0024]FIGS. 12a-b. c-ret expression is sufficient to mediate binding andbiological responses to GDNF in fibroblasts. (a) Iodinated GDNF could becross linked to 3T3 cells stably transfected with MEN2a-ret or wild typec-ret expression plasmids. Untransfected 3T3 cells (3T3) did not bindGDNF. The specificity of the binding was demonstrated by displacement ofthe labeling with 50× excess cold GDNF. (b) GDNF promotes survival andgrowth responses in 3T3 fibroblasts stably transfected with a c-retexpression plasmid. Untransfected cells did not respond to GDNF.

[0025]FIGS. 13a-c. c-ret mRNA expression in adult brain and indeveloping substantia nigra. (a) Ribonuclease protection analysis (RPA)of c-re: mRNA expression in different regions of the adult rat brain.(b) RPA of c-ret mRNA expression during development of the rat ventralmesencephalon (nigra), and of GDNF mRNA expression in the developingstriatum. (c) mRNA expression is indicated in arbitrary units where 100corresponds to the level of expression in the respective regions innewborn animals.

[0026]FIGS. 14a-h. c-RET is expressed in GDNF-responsive substantianigra dopaminergic neuron. (a) Dark field autoradiogram of c-ret mRNAexpression analyzed by in situ hybridization in the adult substantianigra. Scale bar, 40 μm. (b) Bright field autoradiogram showingsubstantia nigra neurons containing c-ret mRNA. Scale bar, 7.5 μm. (c)Immunohistochemical analysis of c-RET protein expression in the adultsubstantia nigra. Scale bar, 27 μm. (d) autoradiogram showing in situhybridization for c-ret mRNA in the adult rat brain after a unilaterallesion with 6-OHDA. The injection of this toxic dopamine analogue in themedial forebrain bundle ensures that only cells which actively take upand retrogradely sport dopamine will be compromised. Note thedisappearance of the labeling for c-ret mRNA in the lesioned substantianigra (arrowhead) 1 day and 5 days following the lesion.(e)Immunohistochemical analysis of c-RET protein expression in the adultsubstantia nigra after lesion with 6-OHDA and grafting of mocktransfected fibroblasts (control graft). Note the nearly completeabsence of c-RET-LI caused by the lesion. Scale bar, 20 μm. (f) Graftingof GDNF-expressing fibroblasts rescues c-RET-LI. Note c-RET positivefibers surrounding and entering the GDNF producing graft (arrows). Samemagnification as in (e). (g) Immunohistochemical analysis of cRETprotein expression in the adult locus coeruleus after lesion with 6-OHDAand grafting of mock transfected fibroblasts (control graft). Scale bar,23 μm. (h) Rescue of cell bodies expressing c-RET-LI by GDNF in of6-OHDA lesioned locus coeruleus. Same magnification as in (g). Graft ison the right in (e) and (f), and above in (g) and (h).

[0027]FIGS. 15a-c. PC12 and NB2/a cells respond to GDNF and bind GDNF.(a) GDNF promotes survival of serum-deprived PC12 cells. (b) GDNFincreases the number of NB2/a cells. (c) ¹²⁵I-GDNF binds to PC12 andNB2/a cells in the absence (open column) or presence (filled column) of50-fold unlabeled GDNF.

[0028]FIG. 16. Affinity crosslinking of ¹²⁵I-GDNF to cell lines.¹²⁵I-GDNF was crosslinked to PC12 cells (lane 1), SY5Y cells (lane 2),E20 rat kidney cells (lane 3) and NB2/a cells (lane 4), and theresulting complexes were precipitated from detergent lysates byanti-GDNF antibodies (Santa Cruz).

[0029]FIGS. 17a-b. GDNF specifically binds to c-RET. ( a) ¹²⁵I-GDNF wascrosslinked to NB2/a cells in the presence (+) or absence (−) of1000-fold excess of unlabeled GDNF (PeproTech EC Ltd.), and theresulting complexes were precipitated from detergent lysates by cocktailof monoclonal and polyclonal (Santa Cruz) anti-c-RET antibodiesrecognizing the extracellular and intrellular domain of cRET,respectively. Lysates were also precipitated by monoclonalanti-neurofilament antibodies 13AA8 (lane 3), by Protein A-Sepharose(lane 4) and by WGA-Agarose (lane 5). (b) ¹²⁵I-GDNF binds to COS cellstransiently expressing c-RET, but not to mock-transfected (with pBK-CNVplasmid) COS cells. Open column represents binding in the presence, andfilled column in the absence of 50-fold excess of unlabeled GDNF.

[0030]FIG. 18. GDNF increases tyrosine phosphorylation of c-RET intransfected COS cells. c-RET was immunoprecipitated from detergentlysates of GDNF-treated (+) (lane 1) or untreated (−) (lane 2) COS cellstransfected (lane 3) with c-ret cDNA or mock-transfected with PBK-CMVplasmid. (a) immunoblot probed with antis-RET antibodies (Santa Cruz).(b) the same filter reprobed with anti-phosphotyrosine antibodies.

[0031]FIGS. 19a-h. GDNF binds in situ to c-ret-positive developingenteric neurons. (a, b) Dark-field (a) and corresponding bright-field(b) microphotographs of GDNF antisense cRNA hybridization to paraffinsections through E15 rat gut. (c, d) Dark-field (c) and correspondingbright-field (d) microphotograph of in situ binding of ¹²⁵I-GDNF to E15rat gut explants. (e) c-ret antisense cRNA hybridization to acryosection through E15 rat gut. (f) Immunostaining of E15 rat gutcryosection with anti-peripherin antibodies. (g) GDNF sense cRNAhybridization to E15 rat gut section. (h) In situ binding of ¹²⁵I-GDNFto E15 rat gut explants in the presence of 250-fold excess of unlabeledGDNF. - - - , muscle layer; n, intestinal nerve plexus. Bar, 100 μm.

[0032]FIGS. 20a-b. Crosslinked GDNF-c-RET-complexes are obtained fromGDNF-responsive cell lines and from c-ret-transfected cells (a)¹²⁵I-GDNF was crosslinked with EDAC to PC12 cells, NB2/a cells,dissociated E20 rat kidney cells, and COS cells, and the resultingcomplexes were precipitated by anti-GDNF antibodies. (b)EDAC-crosslinked ¹²⁵I-GDNF-c-RET complexes were immunoprecipitated withanti-c-RET antibodies from the extracts of PC12 cells, stablyc-ret-transfected (Ret.-3T3) or mock-transfected (mock-3T3) 3T3 cells,as well as from dissociated E15 kidney cells in the presence (+) orabsence (−) of 500-fold excess of unlabeled GDNF or TGF-β1. The ˜50Kbands in all gels are the crosslinked dimers of GDNF.

[0033]FIGS. 21a-b. GDNF increases c-RET autophosphorylation in stablytransfected 3T3 cell line. (a) GDNF dose-dependently increases tyrosinephosphorylation of 160 kD isoform of c-RET in c-ret-transfected(ret-3T3) but not in mock-transfected (mock) cells. (b) GDNFtime-dependently increases tyrosine phosphorylation of 160 kD isoform ofcRET in c-ret-transfected 3T3 cells. Upper panels (Ret.-PTyr) are theimmunoblots stained with anti-phosphotyrosine antibodies, and lowerpanels (Ret.) show the reprobing of the corresponding filters withanti-c-RET antibodies.

[0034]FIG. 22. GDNF increases the number of trkC-3T3 fibroblaststransiently expressing c-RET (open squares), but not mock-transfectedcells (filled squares). c-ret and mock-transfected cells in fiveparallels were treated with rat GDNF at indicated concentrations, orwith NT-3, for five days. Cell number, quantified with Abacus™ CellProliferation Kit (Clontech), is expressed as a percent of the controlcells without growth factors. *, p<0.001 compared to mock transfectedcells.

[0035]FIG. 23a-b. Purification of receptor from L6 myeloblast cells. (a)Plasmon resonance analysis of fractions obtained from anion exchangechromatography of L6 cell lysates. Total protein of fractions is alsodepicted. (b) Further purification of 1M fraction obtained from (a) byhydrophobic interaction chromatography.

[0036]FIG. 24. Autoradiographic film of the ligand blot ¹²⁵I-GDNF withproteins from adult rat brain (lane 2) and liver (lane 3). 50-foldexcess of unlabeled GDNF (lane 1) significantly reduces the binding.

[0037]FIG. 25. Amino acid sequence of GDNFR-β.

[0038]FIGS. 26a-b. Comparison of amino acid sequences of a) rat GDNFR-αand GDNFR-β and b) rat and human GDNFR-α and GDNFR-β.

[0039]FIG. 27. Expression of GDNFR-α mRNA using RNAse protectionanalysis.

[0040]FIG. 28. Expression of GDNFR-β mRNA using RNAse protectionanalysis.

[0041]FIG. 29. Chemical cross linking of ¹²⁵I-GDNF to GDNFR-α andGDNFR-β in transfected COS cells.

[0042]FIG. 30. Comparison of complexes obtained upon cross linking of¹²⁵I-GDNF to GDNFR-α and GDNFR-β in COS transfected cells with complexesobtained upon cross linking of ¹²⁵I-GDNF to receptors on RN33B cells.

[0043]FIG. 31. Effect of co-transfection of GDNFR-α and GDNFR-β withc-Ret in COS cells upon phosphorylation of c-RET (a) and effect oftransfection of GDNFR-α and GDNFR-β in Neuro-2A cells uponphosphorylation of c-RET.

[0044]FIG. 32. The cDNA sequence for rat GDNFR-β.

[0045]FIG. 33. (a) The cDNA sequence of human GDNFR-β. The translationtermination site and the sequences of the primers used to amplify the 5′end of the gene are marked in bold. The first and last 6 nucleotides ofthe sequence (nucleotides 469-1490) derived from I.M.A.G.E. EST clonesare underlined. (b) The alignment of the predicted human and rat GDNFR-βand GDNFR-α proteins. N-glycosylation sites are underlined and aminoacid residues identical in at least three out of the four sequences aremarked in bold.

[0046]FIG. 34. Northern blot showing the expression of GDNFR-β mRNA inmultiple human tissues. The molecular weight marker sizes are the samein all the filters. The lower panels present the same filtersrehybridized with human β-actin probe.

[0047]FIG. 35. Grayscale image of fluorescent in situ hybridization ofthe human (a) and mouse (b) GDNFR-β genes on metaphase chromosomes.

[0048]FIG. 36. GDNFR-β mediates GDNF-induced c-Ret autophosphorylationin transiently transfected COS-7 cells.

[0049]FIG. 37. Bright and dark field images of consecutive sectionsthrough adrenal gland (A, B,C,), kidney (D, E, F), small intestine (G,H, I), spinal cord (J, K, L), and trigeminal ganglia (M, N, O) of E17rat, hybridized with probes to rat GDNFR-β (B, E, H, K, N) or GDNFR-α(C, F, I, L, O). Arrows indicate low GDNFR-β mRNA expression in theundifferentiated mesenchyme of kidney (E) and enteric neurons of gut(H). Abbreviations: ac=adrenal cortex, am=adrenal medulla, drg=dorsalroot ganglion, en=enteric nervous layer, mn=ventral motoneuron column,nt=neural trunk of trigeminal secretory tubules, tg=trigeminal ganglion,u=tip of ureter bud, vr=ventral root of spinal cord. Bar=200 μm.

DETAILED DESCRIPTION

[0050] A prerequisite for the understanding of the full range andmechanisms of action of GDNF is the characterization of GDNF receptorsand their signaling pathways. Although receptors for other members ofthe TGF-β superfamily are well characterized, GDNF receptors remainedundefined until this disclosure. Disclosed herein is the biochemicalcharacterization of GDNF receptors and their downstream responses insympathetic neurons and responsive cell lines. Using affinity labeling,multiple GDNF binding subunits that mediate cooperative binding of GDNFto embryonic sympathetic neurons are identified. Screening of overthirty cell lines initially revealed high expression of GDNF bindingproteins of 55 kD, 70 kD, 135 kD and 300 kD in conditionallyimmortalized neuronal precursors from the raphe nucleus. As the datademonstrate, GDNF receptors were highly induced after neuronaldifferentiation of these cells, which then became sensitive to thesurvival-promoting effects of GDNF. Different combinations of thesesubunits were also seen in glioma, myoblast and Sertoli cells. Adifferent receptor pattern was found in a motor neuron hybrid cell line,where the predominant component was a CPI-anchored protein of 155 kD.

[0051] Despite the striking similarity with the receptor pattern ofother TGF-β superfamily members, immunoprecipitation experimentsindicated that GDNF receptor subunits of 55 kD, 70 kD, 135 kD, and 300kD are novel proteins. The 55 kD binding protein has been confirmed tobe GDNFR-α described as being cloned from the substantia nigra (Treanoret al. Nature, 382:80-83, 1995) and from the retina (Jing et al. Cell,85:1113-1124,1996). GDNFR-α is attached to the cell membrane by aglycosylphosphatidyl inositol linkage (GPI) and, thus, cannot transmitintracellular signals on its own. The 70 kD binding protein has now beencloned and sequenced from the rat and human and has been designated as“GDNFR-β.”

[0052] We assign the locus of GDNFR-β to human chromosomes 8p21-22 andmouse chromosome 14D3-E1. The 155 kD subunit was subsequently determinedto be the product of the c-ret proto-oncogene, c-RET, a receptortyrosine kinase crucial for the development of parts of the excretoryand nervous systems. GDNF stimulated ERK tyrosine phosphorylation andc-fos mRNA expression with different time-courses in raphe nucleus andmotor neuron cell lines, suggesting that different complements of GDNFreceptor subunits can form distinct signaling complexes.

[0053] Concomitantly, c-RET was identified as receptor for GDNF onadditional cell lines. GDNF rescues c-RET-positive dopaminergic andnoradrenergic neurons in lesion models of Parkinson's disease,suggesting that cRET may mediate the anti-Parkinsonian effects of GDNFin the adult brain.

[0054] c-ret proto-oncogene (Takahashi et al. (1985) Cell, 42, 581-588)encodes a protein that is structurally related to receptor tyrosinekinases (Takahashi et al. (1988) Oncogene, 3,571-578). Its extracellularpart contains an unusual cadherin-like domain and also a cysteine-richdomain, the biological roles for which are not understood. Byalternative splicing, several isoforms of c-ret mRNA have been described(Tahira et al. (1990) Oncogene, 5, 97-102; Myers et al., (1995)Oncogene, 11, 2039-2045;Lorenzo et al. (1995) Oncogene, 10, 1377-1383),but their biological meaning is currently not understood. In severalcell lines, c-ret-encoded proteins with molecular weights of 160 kD and140 kD are described, representing the fully and partially glycosylatedisoforms of 120 kD core protein, respectively (Takahashi et al., 1988).As with other receptor tyrosine kinases, c-RET is activated byhomodimerization followed by phosphorylation of its tyrosine residues.

[0055] In the excretory system, c-ret is expressed in the nephric duct,the ureteric bud and the growing tips of the collecting ducts (Pachniset al., (1993), supra). Mice homozygous for a null mutation in the c-retgene die soon after birth, with kidneys either absent or rudimentary anddisplaying severe defects in the enteric nervous system (Schuchardt etal., Nature 367, 380-3 (1994). Based on this evidence, it had beenproposed that the cognate c-ret ligand may be a growth factor importantfor morphogenesis and neurogenesis.

[0056] During murine embryogenesis, c-ret mRNA is expressed primarily inthe nervous and excretory systems. c-ret mRNA is found in dorsal root,sympathetic, enteric and cranial ganglia (Pachnis et al., Development119, 1005-17 (1993), as well as in post migratory neural crest cells andin various tumors of neural crest origin, including pheochromocytoma,medullary thyroid carcinoma and neuroblastoma (Ikeda, I., et al.Oncogene 5, 1291-6 (1990); Santoro, M., et al. Oncogene 5, 1595-1598(1990). In the developing central nervous system, sites of c-retexpression include the ventral portion of the neural tube, the retinaand motor neurons in spinal cord and hindbrain (Pachnis et al., (1993),supra). However, the pattern of expression of c-ret in the adult nervoussystem has not previously been reported.

[0057] The absence of a known ligand for c-RET has basically hamperedthe studies of intracellular pathways that c-RET can mediate.Comparative analysis of the growth-promoting activity of the epidermalgrowth factor receptor/c-RET chimera expressed in fibroblastic orhematopoietic cells revealed a biological phenotype clearlydistinguishable from that of epidermal growth factor receptor (Santoroet al. (1994) Mol. Cell. Biol. 14, 663-675). We disclose herein thatboth NGF and GDNF promote survival of PC12 cells, whereas only NGFinduces their differentiation, suggesting only a partial overlap in thesignaling pathways of c-RET and trkA, a receptor for NGF. Binding of anadaptor protein Grb2 to oncogenic forms of c-RET has been demonstrated(Borrello et al. (1994) Oncogene, 9, 1661-1668). However, the details ofthe pathways are completely unknown. Now, having GDNF as a ligand, it ispossible to address the intracellular signaling of c-RET upon GDNFbinding.

[0058] Like c-RET, GDNF is abundantly expressed in the muscle layer ofthe gastrointestinal tract and in the condensing mesenchyme of thekidney (Suvanto et al. (1996) Eur. J. Neurosci., 8, 816-822). Further,as disclosed herein, GDNF specifically binds c-RET-positive cells indeveloping gut, GDNF can be cross linked to c-RET in several cell linesand in developing kidney, GDNF specifically induces tyrosinephosphorylation of c-RET, and ectopical expression of c-RET in 3T3 cellsconfers a biological response of these cells to GDNF. Thus, c-RET isactivated by GDNF and mediates its functions.

[0059] As disclosed further herein, GDNFR-α and GDNFR-β mediateGDNF-induced c-RET autophosphorylation in transfected cells. Thepresence of the similarly behaving GDNF presenting proteins may lowerthe amount of GDNF needed to activate c-RET. By Northern hybridization,we disclose that the transcript level of human GDNFR-β mRNA is high inthe adult brain, intestine and placenta and in fetal brain, lung andkidney. Studied by in situ hybridization, GDNFR-β mRNA shows in E17 ratembryo different distribution than that of GDNFR-α mRNA, especially inadrenal gland, kidney and gut. In the developing nervous system, GDNFR-βmRNA expression is restricted to certain neuronal populations, whileGDNFR-α mRNA is widely expressed also in non-neuronal cells. Thedistinct tissue distribution of GDNFR-β mRNA and its ability to mediateGDNF signal in transfected cells suggest a role in signal transductionof GDNF and, possibly, related neurotrophic factors in vivo. The factthat GDNFR-β mRNA is present in some organs (such as adrenal cortex)where GDNF is not available points out to a possibility that some otherligand (like GDNF homolog such as neurturin and persefin; Kotzbauer etal., Nature, 384:467470, 1996 and Kotzbauer et al., Differentiation andDegeneration, Keystone Symposia on Molecular and Cellular Biology, Taos,N. Mex., Mar. 27-Apr. 2, 1996, p. 136, respectively) may use GDNFR-β intheir signal transduction. Likewise GDNFR-β may also be used in theactivation of other signaling receptors than Ret.

[0060] GDNF and the genes responsible for its signal transduction are ofgreat clinical interest due to their potential use in therapy formotoneuron and Parkinson's diseases. In addition, these genes areintensively studied as possible candidate disease genes for congenitalor inherited disorders affecting the survival of the neurons insubstantia nigra and in the gastrointestinal tract.

[0061] The product of the c-ret proto-oncogene plays important roles inhuman disease. Rearrangements and mutations in the c-ret gene areassociated with several tumors e.g. familial medullary thyroidcarcinoma, multiple endocrine neoplasia type 2, etc., but also withHirschsprung disease, a disorder that is characterized by the absence ofenteric neurons in the hindgut, resulting in obstipation and megacolonin infants and adults (reviewed in Mak, Y. F. and Ponder, B. A. J.(1996)Curr. Op. Genet. Dev., 6, 82-86). Identification of GDNF as aligand for c-RET further enables the analysis of the molecular basis ofthese diseases. Particularly, the mutations in GDNF gene can now bestudied as possible cause for the Hirschsprung disease in the caseswhere c-ret locus is not mutated.

[0062] At present, there is no candidate disease assigned to the humanlocus of GDNFR-β gene i.e., 8p21-22, but since it probably participatesin the signal transduction of GDNF in neurons, the new receptor GDNFR-βis likely to be of great interest in investigations concerningneurodegenerative diseases. In addition, the gene for GDNF-β is a potentcandidate disease gene for congenital disorders that resemble thephenotypes of GDNF or RET Knock-out mice (e.g. Hirschsprung disease,kidney aplasia and dysplasis), and we are screening for mutations inthese developmental orders.

[0063] The phrases “GDNF receptor” and “receptor for GDNF” as usedherein each refer to a single subunit which binds GDNF as well ascombinations of the receptor subunits which bind GDNF.

[0064] The term “effect” as used herein means an alteration or change.An effect can be positive, such as causing an increase in some material,or negative, e.g., antagonistic or inhibiting.

[0065] The term “homolog” as used herein refers to a compound orcomposition having a similar biological effects as GDNF, such as aredisclosed herein.

[0066] The term “analog” as used herein refers to a compound orcomposition having an antagonistic effect on the biological effects ofGDNF.

[0067] The term “isolated” as used herein in reference to a GDNFreceptor means a compound which has been separated from its nativeenvironment or, if recombinantly expressed, from its expressionenvironment.

[0068] The phrase “substantially pure” as used herein in reference to acompound means an isolated compound which has been separated from othercomponents which naturally accompany it. Typically, a compound issubstantially pure when it is at least 75%, more preferably at least90%, and most preferably 99% of the total material as measured, forexample, by volume, by wet or dry weight, or by mole percent or molefraction.

[0069] The phrase “non-permissive culture conditions” as used hereinrefers to conditions which do not normally support survival of the cellsbeing cultured in vitro, e.g., temperature, media components, etc.

[0070] The phrase “an excess” as used in reference to the addition oflabeled GDNF in a competitive assay refers to an amount of labeled GDNFsufficient to facilitate the detection of a competing compound—forexample, an amount of labeled GDNF which is twice the amount of thecompound to be tested.

[0071] The term “bind” as used herein refers to the interaction betweenthe GDNF ligand and its receptor, the binding being of a sufficientstrength and for a sufficient time to allow the detection of saidbinding under the conditions of the assays disclosed herein.

[0072] The term “about” in reference to a numerical value means ±10% ofthe numerical value, more preferably ±5%, most preferably ±2%.

[0073] Any claims to sequences herein encompass those insubstantialalterations which can be made to a sequence without effecting function,i.e., substantially the same sequences. For example, a change in anucleotide within a codon that results in the same amino acid asoriginally encoded by the codon is “substantially the same sequence.”Also, a conservative amino acid substitution within the sequence thatdoes not affect function is also “substantially the same sequence.”

[0074] The specific examples presented below demonstrate that:

[0075] 1) GDNF receptor is present in multiple neuronal and non-neuronalcell types;

[0076] 2) GDNF receptor is composed of multiple subunits which cooperateto achieve high affinity binding;

[0077] 3) members of the ERK/MAP kinase family are components of theGDNF signaling mechanism; and

[0078] 4) c-RET is a functional receptor for GDNF.

1. Novel GDNF Receptor Expression in Multiple Neuronal and Non-NeuronalCell Types

[0079] Heretofore unidentified receptors are identified herein as GDNFreceptors. These novel GDNF receptors were found in cells lines ofdifferent origins, although they appeared to be most abundant inneuronal cells. Preferably, the cell lines are selected from the groupconsisting of RN33B, RN46A, and C6 (see Table I), with RN33B being mostpreferred. The identification of GDNF receptors in many of these celltypes suggests novel cellular populations responsive to GDNF in vivo.GDNF has been shown to promote survival and phenotype of distinctsubpopulations of neurons, in particular dopaminergic and noradrenergiccentral neurons, as well as spinal and facial motor neurons. Given theactivities of GDNF in various monoaminergic neurons, the discovery ofGDNF receptors in cell lines derived from the medullary raphe indicatethat serotonergic neurons may also respond to GDNF in vivo. Theendogenous expression of GDNF by these cells suggests that this factormay act in a paracrine/autocrine fashion within the raphe nucleus.Expression of GDNF receptors in Sertoli TM4 cells suggests non-neuronalroles for GDNF in developing testis. In vivo, the temporal expression ofGDNF mRNA in testis correlates with the expansion of the Sertoli cellpopulation (Trupp et al., supra) which, together with the discovery ofGDNF receptors on the TM4 cell line, suggest an autocrine action of GDNFduring Sertoli cell maturation. Similarly, the presence of GDNFreceptors in rat myoblast L6 cells, together with the expression of indeveloping muscle in vivo (Henderson et al., supra; Trupp et al.,supra), indicates a potential paracrine role of GDNF during myogenesis.Despite the presence of receptors and biological activities of GDNF onembryonic sympathetic neurons, PC12 cells which had been differentiatedinto sympathetic-like neurons with NGF did not express GDNF receptorsunder initial experimental conditions. As discussed below, however, GDNFreceptors were ultimately identified on PC12 cells.

[0080] GDNF receptors are absent in the pons noradrenergic cell lineCATH.a. Given the robust effects of GDNF on adult central noradrenergicneurons from the locus coeruleus, the absence of GDNF receptors inCATH.a is intriguing. Recently, however, Gong et al. reported that GDNFcan prevent the degeneration of CATH.a cells induced by 6-OH-dopaminetreatment (Gong et al., 21 Abs. Soc. Neurosci., 1789, 1995), suggestingthat GDNF receptors may be induced in these cells after 6-OH-dopaminelesion. Indeed, in vivo studies have shown that GDNF elicits a moreprofound induction of the phenotype of noradrenergic neurons following6-OH-dopamine injection than in the non-lesioned locus coeruleus.Further, Treanor et al. recently reported upregulation of GDNF bindingin sections of the substantia nigra after medical forebrain bundletransaction (Treanor et al., 21 Abs. Soc. Neurosci. 1301, 1995),suggesting that the receptor upregulation may be a general mechanism ofcontrol of GDNF responsiveness in the central nervous system.

[0081] GDNF receptor upregulation was also observed during in vitrodifferentiation of raphe nucleus cells. These lines have recently beenshown to retain the ability to respond to local microenvironmentalsignals after transplantation into the adult brain, where theydifferentiate in a direction that is consistent with that of endogenousneurons in the transplantation site (Shihabuddin et al., 15 J. Neurosci.6666, 1995). In vitro, however, a shift to the non-permissivetemperature differentiates them along default pathways intoglutamatergic (RN33B) or serotonergic (RN46A) phenotypes, respectively.Differentiation in culture has also been shown to upregulate expressionof receptors for other trophic factors in these cells, including theneurotrophin receptors p₇₅ ^(LNGFR) and trkB (Whittemore and White, 615British Res. 27, 1993). Although they can give rise to differentneuronal types depending upon the site of transplantation, RN33B cellsare not able to generate glial elements, suggesting these cellsrepresent neuronally restricted multipotent precursors (Shihabuddin etal., supra). In this respect, it is interesting to note the absence ofGDNF receptors in two pluripotent neuronal stem cell types (Renfranz etal., 66 Cell 713, 1991; Snyder et al., 30 68 Cell 33, 1992) suggestingthat these cells are less restricted than the raphe nucleus cell lines.Taken together, these observations suggest that GDNF receptor expressionmay initially appear in newly differentiated post-mitotic neurons andincrease progressively during neuronal maturation.

2. Multiple GDNF Receptor Subunits

[0082] The data demonstrate that novel GDNF receptor is composed ofmultiple subunits which cooperate to achieve high affinity binding. Thecooperative binding of GDNF to embryonic sympathetic neurons may thus bean indication of a multi-step mechanism of receptor assembly. Becausebinding assays were performed at 4° C., binding cooperativity isunlikely to have resulted from substantial lateral mobility oftransmembrane receptor proteins, suggesting that GDNF binding inducesconformational changes on receptor complexes that are partiallypreformed on the membrane. The nearly identical affinities of thedifferent GDNF receptor subunits obtained by cross linking also supportthe notion of cooperative binding of GDNF to a partially pre-assembledreceptor complex.

[0083] The structural similarities between GDNF and members of the TGF-βsuperfamily suggest that receptors for GDNF might conform to some of theprototypes described for receptors of members of the TGF-β family.Indeed, the pattern of GDNF binding proteins described herein isstrongly reminiscent of type I, type II and type III TGF-β receptors.

[0084] Despite the overall similarities between GDNF and TGF-βsuperfamily receptors, no GDNF receptors could be detected in severalcell lines known to express various TGF-β and activin receptor subunits,including the mink lung epithelial cell line MvILu. In agreement withthis observation, no binding of GDNF has been detected in COS cellstransfected with different combinations of known type I and type IITGF-β superfamily receptors (Ibanez, C., unpublished; P. ten Dijke,personal communication), including the recently isolated type IIreceptor for BMPs (Rosenzweig et al., 92 PNAS USA 7632, 1995) and anovel brain-specific type I receptor (Ryden et al., 21 Abs. Soc.Neurosci 1754, 1995). Moreover, no GDNF receptor complexes could berecovered after immunoprecipitation with antipeptide antisera againstany of the cloned TGF-β superfamily receptors, indicating that GDNFreceptor components are novel proteins.

3. c-RET is a Receptor for GDNF

[0085] GDNF receptors were found in a motor neuron-neuroblastoma hybridcell line, but not in a basal forebrain cell which was also a hybridwith the same neuroblastoma, suggesting that the receptors detected onMN-1 cells represent physiologically relevant motor neuron GDNFreceptors. In contrast to raphe nucleus cells, GDNF expression could notbe detected in the motor neuron cell line, consistent with atarget-derived mode of action for muscle-derived GDNF in vivo (Hendersonet al., 266 Science 1062, 1994; Trupp et al., supra). GDNF binds to andinduces tyrosine phosphorylation of the these receptor which wereidentified as the product of c-ret. c-ret was also able to mediate GDNFbinding and survival/growth responses to GDNF upon transfection intonaive fibroblasts. Moreover, dopaminergic neurons of the adultsubstantia nigra were found to express high levels of c-ret mRNA, andc-RET expressing dopaminergic and noradrenergic neurons in the CNSresponded to the protective effects of exogenous GDNF in vivo. Together,these data indicate that the product of the c-ret proto-oncogene encodesa functional receptor for GDNF which may mediate the neurotrophiceffects of this factor on dopaminergic, noradrenergic and motor neurons.

[0086] The results disclosed herein indicate that the c-RET receptortyrosine kinase is a signal transducing receptor for GDNF. This findingis surprising, given that all receptors for members of the TGF-βsuperfamily characterized so far are receptor serine-threonine kinases(Derynck, R. Trends Biochem Sci 19, 548-553 (1994); Attisano et al.,J.Bba-Mol Cell Res, 222, 71-80 (1994)). GDNF is in fact a very divergentmember of the TGF-β superfamily, with which it shares primarily thespacing between conserved cysteine residues in the amino acid sequencer.Its ability to interact with a receptor tyrosine kinase indicates afurther functional divergence from other members of the TGF-βsuperfamily. Conversely, these findings could suggest that other TGF-βsuperfamily members may also utilize receptor tyrosine kinases.

[0087] The following results disclosed herein also implicate the c-retproto-oncogene product as a functional receptor for GDNF:

[0088] i) GDNF binds to COS cells ectopically expressing the c-retproto-oncogene;

[0089] ii) GDNF can be chemically crosslinked to the product of thec-ret proto-oncogene ectopically expressed in COS cells or from NB2/aand PC12 cells;

[0090] iii) the c-ret proto-oncogene product ectopically expressed inCOS cells, but also in NB2/a cells, becomes rapidly phosphorylated ontyrosine residues upon GDNF binding;

[0091] iv) GDNF promotes biological effects i.e. mitogenic or trophic incells expressing c-ret proto-oncogenic products.

[0092] GDNF specifically binds to RET-expressing (FIGS. 19c, d, h)enteric neurons and the tips of ureteric buds in developing kidney.These tissues were absent or severely reduced in c-ret-deficient mice(Schuchardt et al. (1994) Nature, 367,380-383; Durbec et al. (1996)Development, 122, 349-358). The data disclosed herein furtherdemonstrate GDNF-c-RET complexes from GDNF-responsive andc-ret-transfected cells and from embryonic kidney cells. Finally, GDNFtime and dose-dependently activates c-RET, and introduction of c-retinto GDNF-nonresponsive cells results in GDNF-responsiveness.

4. Downstream Signaling Pathways Activated by GDNF Receptor

[0093] Investigation of GDNF signal transducing mechanisms in raphenucleus and motor neuron cell lines has been conducted. The downstreamresponses elicited by GDNF in these cells demonstrate that the GDNFbinding proteins identified herein represent functional GDNF receptors.The initial biochemical characterization of GDNF signal transductionpathways has identified members of the ERK/MAP kinase family ascomponents of the GDNF signaling mechanism. ERK/MAP kinase activation byphosphorylation is the final step in a cascade of kinases that is set inmotion after activation of the Ras pathway by various growth factors,including TGF-β (Yan et al., 269 J. Biol. Chem. 13231, 1994; Hartsoughand Mulder, 270 J. Biol. Chem. 7117, 1995) and nerve growth factor(Thomas et al., 68 Cell 1031, 1992; Wood et al., 68 Cell 10 II, 1992).More recently, ERK2 has also been shown to form part of the signaltransduction pathway activated by several cytokines, such as interferonsand interleukins, which are not known to activate Ras (David et al., 269Science 1721, 1995). Whether or not Ras activation is one of the stepsin the signaling transduction mechanism of GDNF is an area of furtherinterest.

[0094] Interesting differences were found between the patterns of ERKphosphorylation induced by GDNF in raphe nucleus RN33B cells and inmotor neuron MN-1 cells. GDNF treatment stimulated very rapid (maximumat 5 min) and transient (undetectable after 60 min) tyrosinephosphorylation of ERK1 and ERK2 in RN33B cells, but relatively slower(maximum at 15 min) and more sustained (still detectable after 120 min)phosphorylation of ERK2, but not ERK1, in MN-1 cells. That thesedifferences may have functional significance is suggested by recentobservations made in PC12 cells treated with different growth factors.Exposure of PC12 cells to NGF or fibroblast growth factor (FGF) resultsin neuronal differentiation and in sustained elevation of Ras activityand ERK tyrosine phosphorylation (Qiu and Green, 7 Neuron 977, 1991). Incontrast, treatment with epidermal growth factor, which stimulates DNAsynthesis and proliferation of PC12 cells, results in only transient (<1hr) activation of Ras and ERKs (Qiu and Green, 1991). Thus, differenttime-courses of ERK activation underlie different biological responsesin PC12 cells. Taken together, the different patterns of GDNF receptorsand GDNF-induced ERK phosphorylation in RN33B and MN-1 cells suggestthat different GDNF receptor subunits can cooperate to assemble distinctsignaling complexes in different cell types. Whether different GDNFsignal transduction pathways underlie the different biological effectsof GDNF is an area of further interest.

[0095] Upon activation, ERKs translocate to the nucleus where theyphosphorylate and thereby regulate the activity of transcription factorswhich, in turn, control gene expression. Phosphorylation of p67^(SRF)and p62^(TCF) transcription factors recruits them to the serum responseelement (SRE) in the c-fos gene promoter and stimulates c-fos genetranscription (Gille et al., 358 Nature 414, 1992). Transcription ofc-fos is rapidly and transiently induced after various stimuli,including exposure of PC12 cells to NGF (Millbrandt, 83 PNAS USA 4789,1986) and of osteoblastic cells to TGF-β (Machwate et al., 9Mol.Endocrin. 187, 1995).

[0096] c-fos forms part of the AP-1 transcription factor complex, whichis thought to be involved in the regulation of multiple genes, includinggrowth factor, neuropeptide and neurotransmitter synthesizing enzymegenes (Gizang-Ginsberg and Ziff, 4 Genes Dev. 477, 1990); Hengerer etal., 87 PNAS USA 3899, 1990; Jalava and Mai, 9 Oncogene 2369, 1994). Thestimulation of c-fos transcription by GDNF indicates a role for AP-1complexes in GDNF-induced gene expression. Thus, c-fos could mediate theincrease in the tyrosine hydroxylase (TH) expression observed upon GDNFtreatment of central noradrenergic neurons, or the GDNF-inducedupregulation of vasoactive intestinal peptide (VIP) andpreprotachykinin-A (PPTA) mRNAs in cultured sympathetic neurons from thesuperior cervical ganglion (Trupp et al., 130 J. Cell Biol. 137, 1995).

[0097] The effects of GDNF on the survival of differentiatedserotonergic raphe nucleus cells indicate that the GDNF receptorsidentified on these cells are able to elicit relevant biologicalresponses. The fact that cessation of proliferation, differentiation,and GDNF responsiveness were concomitant with increased GDNF receptorexpression in these cells, suggests that GDNF may be a survival factorfor developing serotonergic raphe neurons in vivo. The data of thispatent disclosure suggest a role for ERKs and c-fos in GDNF-mediatedneuron survival. This can be directly established using dominantnegatives or antisense oligonucleotides.

[0098] The GDNF receptor subunits and complexes disclosed herein havewide-range applicability. The identification and isolation of GDNFreceptor facilitates rational drug design for drugs useful in treating,for example, neuronal disorders, particularly those involving neuronalcell death. As was discussed previously, GDNF has been shown to promotesurvival of adult substantia nigra neurons in vivo followingpharmacological treatments and lesions that mimic Parkinsoniansyndromes, as well as survival responses in other neuronal cell lines.The drugs can be tested for binding affinity to GDNF receptor, and fortheir influence on the downstream effect of GDNF disclosed below—i.e.,the phosphorylation of ERK2 and ERK1. As GDNF receptor has also beenidentified on malignant cell lines, design of drugs for use in cancertherapy is also evident. Further, considering structural similarity withBMP, the development of drugs to be used in treating bone-relateddiseases, i.e., osteoporosis, and for promoting the healing of fracturesis also contemplated.

[0099] Accordingly, isolated receptors according to the presentinvention can be used, inter alia, to screen for compounds orcompositions which are analogs and homologs of GDNF. The potentialanalogs and homologs can be screened initially in competitive bindingassays employing either isolated receptor or cell lines expressing thereceptor—i.e., NB2/a cells—and ¹²⁵I-labeled GDNF. Methods such as thosedisclosed in Example 13 can be used. Analog or homolog activity can thenbe ascertained by further identifying those compounds or compositionswhich, for example, effect a decrease or increase, respectively, in thetyrosine phosphorylation of the RET proto-oncogene. Methods such asthose disclosed in Example 17 can be used. Alternatively, GDNF can beused to screen for and identify other receptors using theabove-reference procedures, or variations thereof.

[0100] The isolation of GDNF receptor also facilitates the developmentof antibodies, both polyclonal and monoclonal, against the receptor.These antibodies can be used to purify the receptors themselves,identify other cells expressing GDNF receptor, thereby prompting othertherapeutic applications, identify other Type I-interactive receptors,as well as be used as drugs themselves. The antibodies can initially beproduced using the ligand/receptor complexes disclosed herein as theimmunogens. Antibodies specific for the ligand can be eliminated fromthe polyclonal serum by absorption with the ligand. Hybridomas formonoclonal production can be selected on the basis of binding of ligand,with the expansion of only those clones which do not bind the liganduncomplexed with the receptor. The antibodies can be prepared by methodswell known to those skilled in the art.

[0101] Alternatively, monoclonal and polyclonal antibodies against GDNFreceptor and GDNF proteins can be used for the characterization and/orisolation of GDNF receptor molecular clones. Further, anti-GDNFantibodies can potentially be used in a screen for homologs, or in theproduction of anti-idiotype antibodies which mimic GDNF.

[0102] The isolation of GDNF receptor also facilitates the isolationand/or production of nucleic acids for the expression of recombinantGDNF receptor, both in vitro and in vivo, for diagnostic and therapeuticapplications. The term “nucleic acids” as used herein includes, forexample, genomic DNA, mRNA, and cDNA. Upon sequencing at least a portionof the GDNF receptor, oligonucleotide primers for isolating genomic DNAfor GDNF receptor and receptor mRNA can be developed.

[0103] cDNA can be prepared from isolated mRNA. The isolation andproduction of nucleic acids can be accomplished utilizing methods wellknown to those skilled in the art using standard molecular biologytechniques such as are set forth in Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982, incorporated herein by reference. Recombinantly producedreceptors can be used in crystallography studies for rational drugdesign. Recombinant extracellular domain can be produced and used as adrug in ligand sink applications, e.g., for ligands with antagonisticproperties.

[0104] The nucleic acids as set forth above can be utilized for genetherapy, using both in vivo and ex vivo techniques. The nucleic acidscan also be used to clone other related receptors using, for example,low stringency screens and reversed transcriptase PCR; and to producecells overexpressing the receptors to screen for other ligands, e.g., bypanning, and other materials serving as receptor agonists, antagonists,or partial agonists and antagonists. Alternatively, recombinantlyproduced receptor itself can be used for the screening assays.Additionally, cells expressing chimeric receptors can be produced usingother TGF-β receptor family members to elucidate signal pathways.Intracellular targets of GDNF receptor can be identified using, forexample, the yeast two-hybrid system. (Chen, et al., 377 Nature 548,1995, incorporated herein by reference.)

[0105] The nucleic acids set forth above can also be used to developtransgenic and/or gene targeted animals. For example, transgenic animalscan be developed for testing the effects of the overexpression of GDNFreceptor. Procedures can be utilized such as are described in Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1986, and Capecchi, M. R.,Trends Genet, 5: 70-76, 1989, both incorporated herein by reference.

[0106] Alternatively, cell lines and transgenic animals unable toexpress GDNF receptor can be prepared to ascertain the effects ofblocking signaling by GDNF. Procedures such as are set forth in Wurst etal., Gene Targeting Vol. 126, edited by A. L. Joyner, IRL Press, OxfordUniversity Press, Oxford, England, pp. 33-61, 1993, incorporated hereinby reference, can be utilized.

[0107] Other applications and modifications are within the spirit andscope of the invention as herein disclosed and will be readily apparentto those skilled in the art.

EXAMPLES

[0108] The following Examples are provided for purposes of elucidationand not limitation on the disclosure or claims.

[0109] Unless otherwise indicated, binding and biochemical studies werecarried out with recombinant rat GDNF produced in Sf21 insect cellsusing a baculovirus expression system. The protein was produced andpurified as previously described (Trupp et al., supra, incorporatedherein by reference). GDNF protein was quantified after silver stainingof SDS/PAGE gels using standard curves obtained with commercial samplesof proteins of molecular weight similar to that of GDNF. Purified humanTGF-β1 was generously provided by Jun-ichi Koumegawa, Kirin Brewery,Tokyo, Japan. Proteins were labeled with Na-¹²⁵I by the chloramine-Tmethod to a specific activity of approximately 1×10⁸ cpm/μg.

[0110] Unless otherwise indicated, binding assays were performed asfollows. Cells were incubated with iodinated GDNF in Dulbecco'sphosphate buffered saline and 2 mg/ml bovine serum albumin (BSA) onMillipore Hydrophilic Durapore 96-well filtration plates. Following twohours of vigorous shaking at 4° C., the cells were washed twice withice-cold binding buffer under vacuum. Dried filters were liberated andbound ¹²⁵I-GDNF quantified in a gamma counter. Non-specific binding wasdetermined by addition of 500-fold excess of cold ligand to the bindingmixtures.

[0111] For affinity labeling, iodinated proteins were bound to monolayercultures of primary neurons or cell lines. Prior to binding, dissociatedchick sympathetic neurons were cultured for 48 hours in the presence ofNGF on polyornithine/laminin coated dishes. Plated cells were incubatedwith 10 ng/ml ¹²I-GDNF at 4° C. in binding buffer as described above.Ligand/receptor complexes were chemically cross-linked for thirtyminutes at room temperature using either disuccinimidyl suberate (DSS)or 1-Ethyl-3(-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC)(Pierce Chemical, Rockland, Ill.). Following quenching of thecross-linking reactions, cells were washed twice with 10 mM Tris/HClbuffered saline, 2 mM EDTA, 10% glycerol, 1% NP40, 1% Triton x-100, 10μg/ml leupeptin, 10 μg/ml antipain, 50 μg/ml aprotinin, 100 μg/mlbenzamidine hydrochloride, 10 μg/ml pepstatin and 1 mM PMSF(proteinaseinhibitors from Sigma). Cleared lysates were boiled for 5 min inSDS/β-mercaptoethanol buffer, fractionated by SDS/PAGE on 4-20% gradientelectrophoresis gels, and visualized by autoradiography. Molecularweights indicated were obtained by subtracting the weight of a GDNFligand monomer, e.g., 25-30 kD, more preferably 23 kD, from theestimated molecular weights of cross-linked complexes visualized bySDS/PAGE. For affinity measurements of cross-linked complexes, cellswere incubated on plates as above in the presence of increasing amountsof unlabeled GDNF. These samples were fractionated by gradient SDS/PAGE,gels were then dried and specific bands excised according to molecularweights determined from autoradiograms, and count in a gamma counter.For immunoprecipitation of affinity labeled receptor complexes, afterbinding and cross-linking with iodinated ligands, cell lysates werecleared and incubated overnight at 4° C. with 5-10 μl of antipeptiderabbit antisera against difference type I, 11 and III TGF-β superfamilyreceptors (ten Dijke et al., 264 Science 101, 1994) (provided by Peterten Dijke, Ludwig Institute for Cancer Research, Uppsala, Sweden).Immunocomplexes were collected with Protein A-Sepharose (Pharmacia,Sweden), washed in lysis buffer and boiled for 5 minutes before SDS/PAGEand autoradiography as above.

Example 1 GDNF Receptors on Embryonic Sympathetic Neurons

[0112] GDNF promotes survival of cultured embryonic chicken sympatheticneurons with similar efficacy and dose response curve as nerve growthfactor (NGF) (Trupp et al., supra). Chicken sympathetic neurons,isolated and prepared as previously described (Trupp et al., supra).Saturation binding with iodinated GDNF was carried out on neuronsisolated from embryonic day 10 (E10) chick paravertebral sympatheticganglia mechanically dissociated in the presence of trypsin. Thepreparation was preplated for two hours on untreated tissue cultureplastic in order to enrich in neurons and allow for re expression ofreceptors. Plots of saturation binding data produced a sigmoidal curvefrom which a Kd of 400 pM could be approximated (FIG. 1a). In agreementwith the sigmoidal behavior of this curve, Scatchard transformation ofthe data produced an inverted U-shaped curve indicative of cooperativebinding (FIG. 1b). The measure of cooperativity of binding can beascertained from a Hill transformation which produced a positive slopeof 1.63 (FIG. 1c), suggesting oligomerization of either ligand orreceptor subunits.

[0113] In order to identify GDNF binding components on the membrane ofsympathetic neurons, chemical cross-linking of ¹²⁵I-GDNF to these cellswas utilized, followed by visualization of the resulting complexes bySDS-PAGE. Gradient gel electrophoresis resolved binding proteins of 70and 300 kD (molecular weights of GDNF receptor subunits reportedhereafter were obtained by subtracting the weight of a GDNF ligandmonomer, e.g., 23 kD, from the estimated molecular weights ofcross-linked complexes visualized by SDS/PAGE), resulting in a patternof bands which resembled that obtained after cross-linking of TGF-β1 toMvILu mink lung epithelial cells (FIG. 2). This result suggested that,like TGF-β receptors, GDNF binding proteins may also form an oligomericreceptor system. A large excess of cold ligand displaced iodinated GDNFfrom the receptor complex indicating the specificity of the labeling.

Example 2 GDNF Receptors on Cell Lines

[0114] Over thirty cell lines were screened for expression of GDNFreceptors using affinity labeling with iodinated GDNF (Table I, infra).Except as otherwise noted, all cell lines used in this study areavailable from and described by the American Type Culture Collection,Rockville, Md. A875 human neuroblastoma was provided by Mart Saarma,University of Helsinki, Finland. CATH.A, a noradrenergic cell lineisolated from a tumor in the pons of transgenic mice expressing SV40 Tantigen under the transcriptional control of a tyrosine hydroxylasepromoter (Suri et al., 1993), was generated and provided by DonaChikaraishi, Tufts University School of Medicine, Boston, Mass. The ratneural stem cell line C17-2 (Snyder et al., 68 Cell 33, 1992) wasgenerated and provided by Evan Snyder, Harvard medical School, Boston,Mass. LAN5 human neuroblastoma was provided by Sven Pahlman, UppsalaUniversity, Sweden. David Hammond, University of Chicago, produced andprovided SN6 cells, a hybrid of mouse basal forebrain cholinergicneurons and the mouse neuroblastoma N18TG2 (Hammond et al., 1986). Humanneuroblastoma SY5Y was a provided by David Kaplan, ABL-Basic ResearchProgram, NCI-Frederick Cancer Research and Development Center,Frederick, Md. ST15A rat neural stem cell line was kindly provided byRon McKay, National Instituted of Health, Md. The generation andcharacterization of raphe nucleus cell lines RN33B and RN46A has beendescribed elsewhere (Whittemore and White, 1993). RN33B and RN46A cellswere obtained from Dr. Scott Whittemore of the University of Miami. Themotor neuron hybrid cell line 2FI.10.14 (referred to here as MN-1) hasbeen previously described (Salazar-Grueso et al., 2 Neuroreport 505,1991).

[0115] Multiple GDNF receptor subunits were detected in various glial,neuronal and non-neuronal cells (Table I). A large molecular weight bandof 300 kD appeared to be the most prevalent species in several celllines after cross linking with disuccymidyl suberate (DSS), and it wasthe only receptor which appeared to bind ligand in the absence of allother receptors (Table 1). A similar pattern was seen in rat C6 glioma,mouse Sertoli TM4 cells, and in two cell lines derived from embryonicneuronal precursors of the rat raphe nucleus, which have previously beenshown to express multiple neuronal markers, including glutamate-(RN33B)and serotonin-(RN46A) synthesizing enzymes (Whittemore and White, 615Brain Res. 27, 1993; White et al., 14 J. Neuro., 1994; Eaton et al., 170Dev. Biol. 169,1995). The consensus pattern in these cells aftercross-linking with DSS consisted of the large molecular weight band of300 kD, and two other receptor subunits with molecular weights at 50-55kD and 65-70 kD, respectively (FIG. 3 and Table I). The 50-55 kDcomponent often ran as a doublet or triplet. The smeary appearance andheterogeneous range of sizes displayed by the large molecular weightcomponent suggests a post-translational modification, presumablyglycosylation, and appears similar to that previously described for typeIII betaglycan TGF-β receptors. This species was somewhat smaller in thecells derived from the raphe nucleus, which could indicate either adistinct core protein or difference levels of glycosylation.

[0116] GDNF receptors could not be detected in pheochromocytoma PC12cells under the present assay conditions of 4° C., even afterNGF-induced differentiation into a sympathetic neuron-like phenotype(Table I, and data not shown). No or very low GDNF receptor expressioncould be seen in various neuroblastomas, and in two pluripotent neuronalstem cells (Table I). TABLE I CELL LINE DESCRIPTION 55 kD 70 kD 135 kD155 kD 300 kD A875 human melanoma − − − − + Balb.SFME mouse embryoniccell − − − − − CATH.a rat pons noradrenergic − − − − − Mv1Lu mink lungepithelia cell − − − − − COS-7 monkey kidney fibroblast − − − − − C2-C12mouse myoblast − − − − − C6 rat glioma + + + − + C17-2 rat CNS stem cell− − − − − FR-3T3 rat fibroblast − − − − − HELA human cervical − − − − −carcinoma LAN5 human neuroblastoma − − − − + L6 rat myoblast − − + − +MN-1 mouse motor neuron − + − + + NB41A3 TH+ mouse − − − − −neuroblastoma NRK-49F rat kidney fibroblast − − − − − PC12 ratpheochromocytoma − − − − − P19 mouse embryo carcinoma − − − − − RN33Brat raphe nucleus + + + − + (glutamat) RN46A rat raphe nucleus + + + − +(seroton) SK-N-MC human neuroepithelioma − − − − − SK-N-SH DRH+ mouse− + − − + neuroblastoma SN6 mouse basal forebrain − − − − − (cholin)ST15A rat CNS stem cell − − − − + SW1353 human chondrosarcoma − − − − −SY5Y human neuroblastoma − − − − − TM3 mouse Leydig cell − − − − − TM4mouse Sertoli cell + + + − + U138MG human glioblastoma − − − − −

[0117] Presence (+) or absence (−) of specific GDNF receptor complexesin the designated cell line.

[0118] Affinity labeling using the cross-linkerethyl-dimethyl-aminopropyl carbodiimide (EDAC) revealed the presence ofan additional GDNF receptor component of 120-135 kD (FIG. 3), only seenafter very long exposure of gels in DSS cross-linked complexes. LikeDSS, EDAC also cross-linked GDNF to receptors of 50-55 kD and 65-70 kD;the high molecular weight subunit of 300 kD was, however, not asefficiently cross-linked by EDAC (FIG. 3).

[0119] The raphe nucleus cell lines are only conditionally immortalizedand do not show signs of transformation. At the non-permissivetemperature and in defined medium, they stop proliferating anddifferentiate into postmitotic neurons (Whittemore and White, 1993).GDNF binding was greatly increased in RN33B and RN46A cells followingdifferentiation (not shown). The overall pattern and the relativeamounts of GDNF receptor components did not change afterdifferentiation.

[0120] Analysis of GDNF binding proteins on the rat myoblast cell lineL6 revealed a different pattern of receptor subunits marked by theapparent absence of 50-55 kD and 65-70 kD receptors. Only the highmolecular weight component of 200-4 OOAkD could be seen aftercross-linking with DSS (FIG. 3). Cross linking with EDAC, however,readily labeled the 120-135 kD subunit previously seen in C6, TM4 andraphe nucleus cell lines (FIG. 3). As in these other cell lines, thiscomponent also run as doublet in L6 myoblasts.

[0121] A distinct receptor complex was found on an embryonic mousespinal cord motor neuron hybrid cell (FIG. 3). This line was obtained byfusion of E14 mouse spinal cord motor neurons and the N18TG2 mouseneuroblastoma, followed by selection of clones expressing high levels ofcholine acetyltransferase activity (Salazar-Grueso et al., 2 Neuroreport505, 1991). Importantly, SN6, a hybrid cell line of embryonic mousebasal forebrain cholinergic neurons and the same N18TG2 neuroblastoma(Hammond et al., 234 Science 1237, 1986), showed no GDNF receptors(Table I), indicating that the GDNF binding proteins seen on the motorneuron cell (hereafter referred to as MN-1) are likely to represent GDNFreceptor components present in spinal motor neurons. As with the L6myoblasts, the predominant receptor in MN-1 cells was preferentiallycross-linked with EDAC, although in these cells it was a larger proteinof 155 kD (FIG. 3). This was subsequently identified to be a c-RETreceptor (see Example 9 below). MN-1 cells also expressed 65-70 kDbinding proteins and low amounts of the 300 kD receptor (FIG. 3 andTable 1).

[0122] In order to dissect the individual constituent affinities of GDNFreceptor subunits, displacement binding assays were performed, followedby cross-linking and SDS-PAGE. Receptor-ligand complexes were visualizedby autoradiography, cut out from the gel and counted in a gamma counter.The resulting displacement curves indicated a Kd of approximately 0.2 nMfor all components on RN33B and MN-1 cells (FIGS. 4a-c). These data atpresent do not clearly establish whether all GDNF receptor subunitsdisplay similar binding affinities or, whether they are all required toassemble a high affinity receptor complex.

Example 3 Biochemical Characterization of GDNF Receptors

[0123] The overall similarity in the pattern of receptors between GDNFand TGF-β prompted an examination of whether any of the previouslyidentified receptors for TGF-β superfamily members was part of the GDNFreceptor complex. Cross-linked ¹²⁵I-GDNF-receptor complexes fromdifferentiated RN33B cells were subjected to immunoprecipitation withdifferent anti-peptide antisera specific for all cloned TGF-βsuperfamily receptors, including type I receptors (ALK-1 to ALK-6), typeII receptors TBRII, ActRII and BMPRII, the type III receptorsbetaglycan, and endoglin. In a parallel control experiment, ¹²⁵I-TGF-β1was cross-linked to type I, type II and type III receptors on the minklung epithelial cell line MvILu followed by immunoprecipitation withantisera against TBRI (ALK-5), TBRII and betaglycan, respectively.Although type I, type II and type III TGF-β receptors were recovered inthe control experiment, none of the GDNF receptor components indifferentiated RN33B cells could be immunoprecipitated by any of thetested antisera (not shown). These data confirmed that the GDNF receptorsubunits expressed on these cells are novel proteins.

Example 4 Endogenous GDNF Expression in Cell Lines Expressing GDNFReceptors

[0124] Traditional models for the action of neurotrophic factors havedescribed them as target-derived polypeptides that promote survival anddifferentiation of specific neuronal subpopulations. More recently, ithas become evident that neurotrophic factors may also have paracrine andeven autocrine modes of action (Ernfors and Persson, 3 Eur. J. Neurosci.953, 1991; Acheson et al., 374 Nature 450, 1995). Expression of GDNFmRNA in cell lines expressing GDNF receptors was examined. Cells werehomogenized in guanidine isothiocyanate (GITC) and β-mercaptoethanol.RNA extraction and GDNF RNAse protection assay were as previouslydescribed (Trupp et al., supra).

[0125] Unexpectedly, all cell lines, with the exception of the motorneuron line MN-1, expressed substantial levels of GDNF mRNA as assayedby RNAse protection analysis (FIG. 5). The highest GDNF mRNA expressionwas found in cells from raphe nucleus, which showed up to 5-fold higherexpression than postnatal day 1 (P1) kidney, one of the richest sourcesof GDNF mRNA in the developing rat (Trupp et al., supra). Interestingly,upon differentiation of RN33B cells, GDNF mRNA expression decreased toabout 30% of the level in undifferentiated cells (FIG. 5). GDNFtreatment of differentiated RN33B cells did not alter the expression ofGDNF mRNA (FIG. 5) or GDNF receptors (not shown).

[0126] Expression of c-ret mRNA was investigated in RN33B, L6, and MN-1cells, using the RNAse protection assay. Ten micrograms of total RNAfrom the cell lines indicated was analyzed using a riboprobecomplementary to 400 nucleotides of coding sequence from the kinasedomain of the mouse c-ret mRNA. Although high expression was seen inMN-1 cells, no c-ret mRNA was detected in either the RN33B or L6 cells(FIG. 6). These results indicate that a signaling receptor for GDNFother than c-RET must be present in these cells.

Example 5 Activation of the ERK Signal Transduction Pathway in GDNFResponsive Cell Lines

[0127] Whether the GDNF binding proteins characterized in cell lineswere able to form ligand-dependent signaling complexes was alsoinvestigated. Cell monolayers in 10 cm plates were incubated at 37° C.in the presence of 50 ng/ml GDNF for the indicated time periods andimmediately lysed with I ml of ice cold lysis buffer (as above) with theaddition of 1 mM sodium othovanadate. Whole cell lysates werefractionated by SDS-PAGE (10% polyacrylamide) and blotted tonitrocellulose filters. Western blots were probed with ananti-phosphotyrosine antiserum (UBI, Lake Placed, N.Y.), followed byhorseradish peroxidase-conjugated goat anti-mouse IgG and developed withthe ECL Western Detection System (Amersham, UK). For reprobing, blotswere first stripped by a 30 minute incubation at 50° C. in 62.5 mMTris-HCl pH6.7, 100 mM β-mercaptoethanol, 2% sodium dodecyl sulphate.After removal of antibodies, blots were probed with a rabbit polyclonalantisera raised against recombinant rat ERK2 (a gift of Teri Boulton,Regeneron Pharmaceuticals Inc., Tarrytown, N.Y.) which recognizes bothERK1 and ERK2, and developed as above using a horseradishperoxidase-conjugated goat anti-rabbit secondary antibody.

[0128] Because of their distinct patterns of GDNF receptor subunits,intracellular signaling responses were initially characterized in theraphe nucleus cell line RN33B and in the motor neuron cell line MN-1.Changes in the pattern of tyrosine-phosphorylated proteins elicited byGDNF treatment of RN33B or MN-1 cells were investigated. Tyrosinephophorylation is a universal mechanism of regulation of intracellularsignaling proteins that is stimulated by numerous cytokines and growthfactors. RN33B and MN-1 monolayers were exposed to a saturatingconcentration of GDNF (5 ng/ml) for different periods of tine, and totalcell lysates were analysed for tyrosine phosphorylation by SDS/PAGE andWestern blotting with an anti-phosphotyrosine monoclonal antibody. Twoproteins with mobilities corresponding to 42 kD and 44 kD, respectively,were phosphorylated on tyrosine within 5 minutes of GDNF treatment ofRN33B cells (FIG. 7A). A similar result was obtained in differentiatedRN33B cells (not shown) of exposure to GDNF.

[0129] Based on comparison of their size with descriptions of growthfactor-induced protein tyrosine phosphorylation elsewhere (Qiu andGreen, 9 Neuron 705, 1992), the 42 kD and 44 kD species would appear tobe, respectively, p42, k2 and p44, kI, two protein serine-threoninekinases members of the extracellular signal-regulated kinase (ERK, alsotermed microtubule-associated protein kinase) family (Boulton et al., 65Cell 663, 1991). To confirm the identity of these proteins as ERK2 andERK1, respectively, protein blots which had been reacted with theanti-phosphotyrosine antibody were stripped and reprobed with a rabbitpolyclonal antibody raised against recombinant ERK2 that recognizes bothERK1 and ERK2 in protein blots. Comparison of autoradiograms of blotsprobed with the anti-phosphotyrosine antibody and the anti-ERK2 antibodyidentified the p42 and p44 proteins as ERK2 and ERK1, respectively (FIG.7a). Although GDNF treatment of MN-1 cells appeared to only stimulatephosphorylation of ERK2, both ERK1 and ERK2 were present in MN-1 celllysates (FIG. 7b). Thus, GDNF treatment stimulated very rapid andtransient tyrosine phosphorylation of ERK1 and ERK2 in RN33B cells, butrelatively slower and more sustained phosphorylation of ERK2 and MN-1cells.

[0130] Activation of the ERK pathway has previously been shown to inducerapid and transient increase in transcription of immediate early genes,including the c-fos proto-oncogene (Gille et al., 358 Nature 414, 1992).Accordingly, the ability of GDNF to induce c-fos mRNA in differentiatedraphe nucleus RN33B cells and in motor neuron MN-1 cells wasinvestigated. For analysis of c-fos mRNA expression in cell lines,culture medium was changed 90 minutes prior to addition of 100 ng/mlGDNF to cell monolayers. At the indicated time intervals, media wasremoved, cells solubilized with guanidine isothiocyanate andβ-mercaptoethanol and RNA extracted as previously described (Trupp etal., supra). Twenty micrograms of total RNA was fractionated on 1%agarose gels containing 0.7% formaldehyde and transferred to Hybond-Cmembranes (Amersham, UK). Northern blots were hybridized with ana-³²P-dCTP labeled rat c-fos gene fragment (Curran et al., 2 Oncogene79, 1987), washed at high stringency and visualized by autoradiographyon x-ray films.

[0131] Cell monolayers were exposed to saturating concentrations of GDNFfor different periods of time and levels of c-fos mRNA were subsequentlyanalyzed in Northern blots of total RNA (FIG. 8). This analysis revealedtransient upregulation of c-fos mRNA 15 minutes after exposure of RN33Bcells to GDNF, return to basal levels 45 minutes after treatment (FIG.8a). c-fos mRNA was also upregulated in MN-1 cells but not until 30minutes of GDNF treatment (FIG. 8b). Elevated c-fos mRNA levelspersisted for about an hour and returned to basal levels 120 minutesafter the initiation of treatment (FIG. 8b). Thus, like tyrosinephosphorylation of ERKS, c-fos mRNA upregulation induced by GDNFtreatment was very rapid and transient in RN33B cells, but somewhatslower in MN-1 cells.

Example 6 Survival Responses Promoted by GDNF in Differentiated RapheNucleus Cells

[0132] Advantage was taken of the conditional nature of theimmortalization of the raphe nucleus serotonergic cell line RN46A byexamining whether GDNF may be a survival factor for differentiated raphenucleus neurons. Survival assays were performed as previously described(Eaton et al., 1995, supra). Briefly, 10⁵ RN cells were seeded tocollagen/fibronectin coated 8-well glass slides and incubated at 33° C.(growth permissive temperature) until 75-90% confluent. The slides werethen shifted to 39° (non-permissive temperature) and serum containingmedium was replaced by B16 defined medium (Brewer and Cotman, 494 BrainRes. 65,1989) containing 1% BSA, 1 μg/ml transferrin, 5 μg/ml insulin,100 mM putrescine, and nM progesterone plus or minus 0-50 ng/ml rhGDNF(Promega, Madison, Wis.). Media and GDNF were replaced every two daysfor 8 days after which the cells were fixed in 4% paraformaldehyde/2%glutaraldehyde, rinsed and coated with a glycerol mounting mediumcontaining 1 mM bisbenzamide (Hoechst dye 33342) to stain viable nuclei.Fields of cells were magnified to 40× on a Zeiss Axiophot microscope,examined for fluorescent nuclei (at 355 nM excitation, 465 nM emission),the images video captured, and the cells counted with Imade I™ software.For each condition, 10 fields of cells were counted from each of 3independent experiments.

[0133] RN46A cells were cultured at the non-permissive temperature indefined medium in the presence of increasing concentrations of GDNF.Nine days after plating, surviving cells were counted and compared withcultures established in the absence of GDNF. A 3-fold increase in thenumber of surviving cells was observed in cultures grown in the presenceof GDNF (FIG. 9). The effect of GDNF on the survival of differentiatedRN46A cells was dose dependent, with an EC50 at 5 ng/ml.

Example 7 Generation, Cloning and characterization of Anti-GDNFMonoclonal Antibodies Immunisation

[0134] Five young female mice were immunised with 35 ug of insectcell-derived recombinant GDNF emulsified with complete Freund's adjuvant(FA). Second and third immunizations were performed 2 and 4 weeks afterthe first one in incomplete FA. All the injections were givenintraperitoneally (i.p.). Two weeks after the last Immunisation,antibody titer in serum was checked by ELISA and Western Blot analysisusing standard methods. The mouse with the highest titer (more than1:2000) was boosted i.p. with 3 μg of GDNF in incomplete FA 3 daysbefore the cell fusion.

Cell Fusion

[0135] Cell fusion was done according to the method of Kohler andMilstein (1975), incorporated herein by reference, with somemodifications.

a) Day Before Fusion

[0136] Viable cells from the Sp2/0 murine cell line were adjusted to2×1O⁵ cells/ml with complete DMEM (10% fetal calf serum, 1% L-glutamine,100 U/ml penicillin and 100 ug/streptomycin sulphate).

[0137] Cells from a non-immunised mouse were obtained from theperitoneal cavity by injection of 0.34M sucrose solution. The cells wereresuspended in complete DMEM containing: hypoxanthine 100 μM;aminopterin 0.4M; and thymidine 16 μM, (HAT medium), to 1×10⁵ cells /ml.100 μl of the cell suspension was added to the 60 inner wells of 96 wellplates and incubated overnight at 37° C. in an atmosphere of 5% C0₂ inair. These cells were the source of growth factors.

b) Fusion

[0138] Spleen cells from the mouse exhibiting the highest serum titer(see above) were homogenized in 10 ml DMEM removing surface fat andother adhering tissue in a sterile hood.

[0139] 4.2×10⁷ Sp2/0 cells were fused with 8.4×10⁷ spleen cells in asolution of melted PEG (3000-3700, Hybri-Max, Sigma). The cells werethen grown in HAT medium at 37° C. in an atmosphere of 5% CO₂ in air.After one week of culture, the wells were inspected. When hybrids cellscovered 10 to 50% of the surface area of the well, the culturesupernatants were assayed for antibody by ELISA.

[0140] For the ELISA, wells of microplates (Costar, EIA/RIA plate highbinding) were coated with 100 ul of 2ug/ml of GDNF diluted incarbonate/bicarbonate buffer, pH 9.6. After an overnight incubation at4° C., the wells were washed with 0.05M phosphate buffered saline, pH7.2, containing 0.05% Tween (PBS-T). Nonspecific binding was blockedwith PBS-T containing 3% non-fatty milk and 1% goat normal serum.Supernatant samples were incubated 4 hours at room temperature.Peroxidase goat anti-mouse antibody was used and the substrate waso-phenylenediamine dihydrochloride (OPD). Plates were read at 492 nm inan ELISA reader. Negative controls included completed medium and normalmouse serum.

[0141] The hybrids were grown in HAT medium up to two weeks afterfusion. Cells were subsequently grown in HT medium until the completionof two cloning procedures, using the limiting dilution method. Aftereach step (when cells reached 10 to 50% confluence), assays for specificantibody in supernatants were done by ELISA. Upon recloning, 5 positivehybridoma clones were chosen and the cells were maintained in completeDMEM for 30 days.

Isotyping of Monoclonal Antibodies

[0142] The class and subclass of the monoclonal antibodies weredetermined by ELISA using a DAKO panel for isotyping of mouse monoclonalantibodies. All five 5 monoclonal antibodies were characterized as IgG₁.

Purification of Monoclonal Antibodies

[0143] Monoclonal antibodies from culture supernatants were purified byProtein G Sepharose fast flow (Pharmacia, Biotech) according tomanufacturer's instructions. Culture supernatants were concentrated andfiltered through a 0.45 μm membrane (Schleicheer and Schull, Germany)and then pumped overnight through the column previously equilibratedwith 2 OmM sodium phosphate, pH 7.0. Ig was eluted with 0.05M glycinebuffer.

Example 8 A Motor Neuron Cell Line Showing Biological and BiochemicalResponses to GDNF

[0144] MN-1 cell monolayers were exposed to increasing concentrations ofGDNF in serum-free medium and assayed 3 days later for cell survival andgrowth by measurement of acid phosphatase activity (Clontech). GDNF wasproduced and purified from baculovirus infected insect cells aspreviously described (Trupp et al.,supra). GDNF treatment of serumdeprived-MN-1 monolayers increased cell number in a dose-dependentmanner (FIG. 10a). The biological response of MN-1 cells correlated withbiochemical and transcriptional responses to GDNF treatment. MN-1 cellmonolayers were exposed to 50 ng/ml GDNF for increasing periods of time,cell lysates were fractionated by SDS/PAGE and Western blots probed withan anti-phosphotyrosine antibody (UBI).

[0145] Several proteins were seen to have increased tyrosinephosphorylation after GDNF treatment of MN1 cells, including a proteinwith an electrophoretic mobility of 42K (FIG. 10b). Based on comparisonof its size with descriptions of growth factor-induced protein tyrosinephosphorylation elsewhere (Boulton, T. G., et al. Cell 65, 663-75(1991), the 42K species would appear to be p42^(erk2), aserine-threonine kinase member of the extracellular signal-regulatedkinase (ERK) family. The identity of this protein as ERK2 was confirmedafter immunoprecipitation with an antiERK2 polyclonal antiserum followedby analysis of tyrosine phosphorylation (FIG. 10c). Lysates ofGDNF-stimulated MN-1 cells were immunoprecipitated with an anti-ERK2antiserum (Santa Cruz) that also recognizes ERK1 followed byantiphosphotyrosine Western blotting. This analysis further revealedthat another member of the ERK family, p44erk1, was also phosphorylatedon tyrosine after GDNF treatment of MN-1 cells (FIG. 10c). Activation ofthe ERK pathway has previously been shown to induce a rapid andtransient increase in the transcription of immediate early genes,including the c-fos proto-oncogene (Gile et al., Nature 358, 414-7(1992).

Example 9 The Product of the c-ret Proto-Oncogene as a SignalTransducing Receptor for GDNF

[0146] GDNF receptor complexes from MN-1 cells could be recovered byimmunoprecipitation with anti-GDNF antibodies or by binding tolectin-Sepharose beads (FIG. 11a). Unexpectedly, the 180 kD receptorcomplex (i.e., c-RET; 180 kD−23 kD=157 kD, which is approximately equalto the 155 kD receptor identified as c-RET—see Example 2, infra) couldalso be recovered by immunoprecipitation with anti-phosphotyrosineantibodies (FIG. 11a), indicating that the GDNF binding protein in thiscomplex could be a receptor tyrosine kinase.

[0147] The product of the c-ret proto-oncogene is highly expressed inprimary motor neurons (Pachnis et al., supra, and Tsuzuki, T., et al.Oncogene 10, 191-8 (1995) and is of similar molecular weight as themajor GDNF receptor component detected in MN-1 cells (Takahashi, M., etal. Oncogene 3, 571-578 (1988). We tested whether this speciesrepresented a C-RET-GDNF cross-linked complex by immunoprecipitationwith anti-c-RET antibodies.

[0148]¹²⁵I-GDNF was cross-linked to MN-1 cells using EDAC and receptorcomplexes were precipitated with antibodies against GDNF (Trupp et al.,supra), lectin Sepharose beads (Formica), anti-phosphotyrosineantibodies (UBI), anti-c-RET antibodies (Santa Cruz) and controlantibodies from non-immune rabbits. An antipeptide c-RET rabbitantiserum readily immunoprecipitated the major 180 kD ligand-receptorcomplex in MN-1 cells (FIG. 11a), while a number of unrelated monoclonaland polyclonal antibodies used as controls failed to immunoprecipitatethis complex (FIG. 11a and data not shown).

[0149] Because the product of the c-ret gene is a receptor tyrosinekinase, we investigated whether GDNF could stimulate tyrosinephosphorylation of the c-RET protein in MN-1 cells. MN-1 cell monolayerswere exposed to GDNF at different concentrations or for differentperiods of time and cell lysates were immunoprecipitated with anti-c-RETantibodies and analyzed by SDS/PAGE and Western blotting withantiphosphotyrosine antibodies as disclosed above. GDNF treatmentstimulated rapid c-RET tyrosine phosphorylation in MN-1 cells (FIG.11b). Maximal phosphorylation was reached 5 minutes after GDNF treatmentand lasted for at least 60 minutes. A dose-response analysis of GDNFinduced c-RET phosphorylation in MN-1 cells showed maximalphosphorylation at 30 ng/ml of GDNF (FIG. 11b), which is similar to theresponse of both serum deprived MN-1 cells (FIG. 10a) and embryonicsympathetic neurons (Trupp et al., supra) to GDNF. Taken together, thesedata indicate that the c-RET receptor may be an important component inthe signal transduction mechanism of GDNF.

Example 10 c-ret Transfection Reconstitutes GDNF Binding and BiologicalActivities to GDNF

[0150] Experiments were conducted to determine whether expression of thec-ret gene product could be sufficient to allow binding of GDNF to cellslacking GDNF receptors. To this end, GDNF binding and cross-linkingexperiments were performed in naive 3T3 fibroblasts, and 3T3 cellsstably transfected with either a wild type c-ret or an oncogenic form ofthis gene found in MEN2a patients (Mulligan et al., Nature, 363:458-460,1993). For c-ret expression in transfected cells, human wild type c-retand MEN2a-ret cDNAs were subcloned in pcDNA3 (Invitrogen). Cold GDNF wasused at 50× molar excess. For survival/growth assays, cells werecultured for 6 days in serum-free medium supplemented with the indicatedconcentrations of GDNF; medium and GDNF were replaced every two days.Cell number was quantified by measurement of acid phosphatase activity(Clontech).

[0151] After immunoprecipitation with c-RET antibodies, GDNF-labeledreceptor complexes of approximately 180K were detected in both MEN2a-retand c-ret transfected 3T3 fibroblasts, but not in untansfected cells(FIG. 12a). The labeling could be displaced by excess cold GDNF,indicating that it represented specific GDNF binding (FIG. 12a).

[0152] Experiments were also conducted to determine whether c-ret couldmediate a biological response to GDNF upon transfection innon-responsive cells. Survival and growth responses to GDNF wereinvestigated in untransfected and c-ret transfected 3T3 fibroblastscultured in serum-free medium. GDNF elicited a dose-dependent increasein cell number in c-ret transfected, but not in untransfected, 3T3 cells(FIG. 12b) which was comparable to the one previously observed inserum-deprived MN-1 cells. Since naive 3T3 cells did not express anyappreciable amount of GDNF receptors prior to transfection (see FIG.12a), it was concluded that c-ret expression was sufficient formediating a biological response to GDNF in these cells.

Example 11 c-ret Expression in Adult Brain and Dopaminergic Neurons ofthe Substantia Nigra

[0153] Experiments were conducted to determine whether the c-ret productmay mediate the neurotrophic effects of GDNF in the brain by examiningthe expression of c-ret in different regions of the rat central nervoussystem. A rat c-ret riboprobe was generated using as template a cDNAfragment obtained by PCR with primers based on sequences U22513 andU22514 (Genbank accession numbers). High levels of c-ret mRNA were foundin MN-1 cells and in rat spinal cord (data not shown). High c-ret mRNAexpression was also found in the adult pons, medulla, locus coeruleusand hypothalamus (FIG. 13a), as well as in thalamus and cerebellum (datanot shown). c-ret mRNA was expressed at barely detectable levels instriatum, hippocampus and cerebral cortex (FIG. 13a). In the ventralmesencephalon, containing the cell bodies of GDNF-responsivedopaminergic neurons, c-ret mRNA levels increased progressively duringpost-natal development (FIG. 13b). A peak of expression was detectedbetween post natal day 6 (P6) and P8, at which time axons ofdopaminergic neurons of the substantia nigra begin innervation of thestriatum, and coincident with an increase in GDNF mRNA expression inthis target region (FIG. 13b). For mRNA quantification, aglyceraldehyde-3-P dehydrogenase (GAPDH) riboprobe was included in theRPA, and values of relative mRNA expression, obtained afterdensitometric scanning of gel autoradiograms, were normalised using theGAPDH signal of each RNA sample. RPA for GDNF mRNA has been previouslydescribed (Trupp et al., supra).

[0154] In situ hybridisation and immunohistochemistry were performed aspreviously described (Arenas, E. & Persson, H. Nature 367, 368-371(1994); Neveu, I & Arenas, E. J. Cell Biol. in press (1996). c-RETprotein was detected using a hamster monoclonal anti-mouse c-RETantibody which also recognises rat c-RET (Lo, supra) followed byfluorescein-conjugated rabbit anti-hamster secondary antibodies(Southern Biotechnologies). In situ hybridization on sections throughthe adult substantia nigra revealed strong labelling over neuronsthroughout this structure (FIGS. 14a-b). In addition, cells positive forc-RET-like immunoreactivity (c-RET-LI) were found throughout the adultsubstantia nigra, with strong labelling over cell bodies (FIG. 14c).

[0155] In order to establish that c-ret expression in the adultsubstantia nigra was confined to dopaminergic neurons, these cells wereselectively lesioned with a unilateral injection of 6-hydroxydopamine(6-OHDA); the cells were then analyzed for c-ret mRNA expression by insitu hybridisation. Lesions of dopaminergic neurons of the substantianigra were performed by stereotaxic injections of 8 pig 6-OHDA in themedial forebrain bundle at the following coordinates: 1.6 mm caudal tobregma, 1.3 mm lateral to midline, and 8.4 mm under the dural surfacewith the incisor bar 5 mm over the interaural line. Animals werepretreated with 25 mg/kg desipramine (i.p.) 30 minutes prior to 6OHDAinjection. 0.75×10⁶ GDNF-expressing fibroblast cells in 3 μl of mediumwere injected supranigrally at the following coordinates: 3,1 mm frominteraural line, 2 mm lateral to midline, and 7 mm under the duralsurface, with the incisor bar at −3.3 mm. Lesion and grafting in thelocus coeruleus were as previously described (Arenas et al., Neuron 15,1465-1473 (1995). The generation and characterisation of GDNF expressingfibroblasts have been described previously (Arenas et al., supra).

[0156] Five hours after the lesion, no difference could be seen betweenipsi and contralateral sides in c-ret mRNA expression (FIG. 14d).However, a marked reduction in c-ret mRNA expression was seen in thelesioned substantia nigra already one day after 6-OHDA treatment, andwas nearly absent 5 days after the lesion (FIG. 14d). c-ret mRNAexpression in the side contralateral to the lesion was, however, notaffected (FIG. 14d). This result indicated that in the adult substantianigra, c-ret mRNA expression was confined to dopaminergic neurons.

Example 12 GDNF Rescues c-RET-Positive Dopaminergic and NoradrenergicNeurons

[0157] Experiments were conducted to determine whether c-RET expressingneurons of the adult substantia nigra and locus coeruleus responded toGDNF. For this, nigral dopaminergic neurons lesioned with 6-OHDA, andwere then examined to determine whether grafts of GDNF expressingfibroblasts induced responses on c-RET immunoreactive neurons. Inlesioned animals that received a graft of control fibroblasts, noc-RET-LI could be detected, indicating a depletion of c-ret-expressingcells by selective lesion of doparinergic neurons in the adultsubstantia nigra (FIG. 14e). However, c-RET-LI could be rescued by theGDNF-expressing graft, where c-RET immunopositive fibers could be seensurrounding and penetrating the graft (FIG. 14f). Similar results wereobtained in the locus coeruleus, where lesion with 6-OHDA depletedc-RET-immunoreactive cell bodies (FIG. 14g), which could be rescued byexogenous administration of GDNF (FIG. 14h). In both brain regions, therescue of c-RET-LI positive cells and sprouting in the animals graftedwith GDNF-expressing fibroblasts paralleled that of tyrosine hydroxylaseimmunoreactivity (data not shown), demonstrating that c-RET-expressingadult dopaminergic and noradrenergic neurons respond to GDNF.

Example 13 Identification of GDNF c-RET Receptors

[0158] PC12 cells and NB2/a cells were washed three times with serumfree RPMI-1640 or DMEM, respectively, plated on noncoated (NB2/a cells)or collagen-coated (PC12 cells) dishes (5000-6000 cells per dish) in thepresence or absence of 50 ng/ml of GDNF (Peprotech EC Ltd.) and thenumber of cells was microscopically counted after 48 hours. PC12 andNB2/a cells were harvested (100,000 cells, five parallels), incubatedwith 10 ng/ml human ¹²⁵I-GDNF (iodinated by Chloramine T method, 100μCi/μg) in the presence or absence of 50-fold unlabeled GDNF for 120-150min on ice, the unbound factor was removed by centrifugation through 30%sucrose cushion, and the cell-associated radioactivity counted on 1271RIAGAMMA counter (IXB Wallac).

[0159] Recombinant human GDNF promoted survival of about 20% of serumdeprived rat pheochromocytoma PC12 cells at concentration of 50 ng/ml(FIG. 15a). Serum-deprived PC12 cells are also maintained by nervegrowth factor (NGF). Upon treatment with (NGF), PCI2 cells also stopdividing and differentiate into sympathetic neuron-like cells with longneurites. Thus, GDNF is a survival-promoting factor for PCI2 cells,although less potent than NGF, but it does not induce differentiation ofPC12 cells at the concentrations studied, presumably because of thedifferences in signal transduction of NGF activated trkA receptors andGDNF receptors.

[0160] Human neuroblastoma NB2/a cells were plated in serum-free mediumin the presence or absence of 50 ng/ml of GDNF and the number of cellswas counted after 48 hr of culture. GDNF significantly increased thenumber of NB2/a cells (FIG. 15b). Monkey COS cells, human SY5Y cells andmouse NIH 3T3 cells showed neither mitogenic nor survival response toGDNF (data not shown). Thus, GDNF exerts biological effects on rat PC12cells and human NB2/a cells, indicating that both cell lines expressfunctional GDNF receptors.

[0161] To determine, whether GDNF binds to the responsive cells, PC12cells and NB2/a cells were incubated with ¹²⁵I-labeled human GDNF at 40°C. as indicated in the legend to FIG. 15. As shown in FIG. 15c, both PC12 and NB2/a cell lines bind GDNF 30 efficiently. More importantly, thebinding of ¹²⁵-labeled GDNF could be competed with a 50-fold excess ofunlabeled GDNF (FIG. 15b). Thus, the binding of GDNF to the receptors onPCI2 and NB2/a cells appears to be specific.

Example 14 Identification of GDNF c-RET Binding Components

[0162] PC12 cells, SY5Y neuroblastoma cells and NB2/a cells wherechemically cross-linked to 125I-GDNF with EDC. 3−5×10⁶ cells ormechanically dissociated cells from 2 E20 rat kidneys were incubatedwith 10 ng/ml of ¹²⁵I-GDNF for I hour on ice and cross linked with 30 mMEDAC (Pierce) for 30 minutes on ice. Detergent lysates wereimmunoprecipitated, the precipitates collected by Protein A-Sepharose,separated on 7% SDS-PAGE, and visualized by Phosphorimager SI (MolecularDynamics).

[0163] The resulting complexes were immunoprecipitated with rabbitantibodies to GDNF, analyzed by SDS-PAGE and visualized byautoradiography. Embryonic kidney cells were also studied as the sourceof putative GDNF receptor (Suvanto, P. et. al., Eur. J. Neurosci., 8,101-107 (1996); Sainio, K. et. al., Nature, (1996) submitted).Cross-linked complexes of 170 and 190 kD were obtained from the extractsof PC12 cells, SY5Y cells and NB2/a cells and a 190 kD complex fromembryonic kidney extracts. (FIG. 16).

[0164] The molecular weights of the cross linked proteins minus GDNF ofapproximately 25-30 kD, substantially, if,not exactly, correspond to themolecular weights of c-RET protooncogene, an orphan receptor tyrosinekinase (Takahashi, M., Ritz, J. & Cooper, G. M. Cell, 42, 581-588, 1985;Takahashi, M. et. al., Oncogene, 3, 571-578 (1988)) (140 kD and 160 kD,representing differently glycosylated forms of c-RET., Tsuzuki, T.,Takahashi, M., Asai, N., Iwashita, T., Matsuyama, M. & Asai, J.Oncogene, 10, 191-198 (1995).

Example 15 Affinity Cross Linking of GDNF to c-RET

[0165] The cross linked complexes were immunoprecipitated from the NB2/acells with the cocktail of antibodies recognizing extracellular andintracellular part of the c-RET receptor. As shown in FIG. 17a (lane 1),the complexes of 170 kD and 190 kD were precipitated by anti-c-RETantibodies, which thus correspond to cross linked GDNFc-RET complexes.Binding of ¹²⁵I-GDNF to c-RET proteins was completely abolished by500-fold excess of unlabeled GDNF (lane 2). No proteins wereprecipitated by monoclonal anti-neurofilament antibodies (lane 3) or byProtein A-Sepharose only (lane 4). No cross linked complexes wereobtained from COS cells (not shown). Since c-ret proto-oncogene is aglycoprotein, ¹²⁵I-labeled NB2/a cell extracts were alsoimmunoprecipitated with wheat germ agglutinin. Again, proteins of 170and 190 kD were obtained (lane 5).

[0166] To establish further that GDNF specifically binds c-RET, themouse c-ret cDNA was cloned into the mammalian expression vector PBK-CMVand transiently expressed in monkey COS cells. Mouse c-ret cDNA(Pachnis, V., Mankoo, B, & Costantini, F. Development, 119, 1005-1017(1993)) in pbluescript SK′ (Stratagene) was cleaved with SaclI and EcoRVand cloned into Sacll and SmaI site of pBK-CNV vector (Strategene). COScells were transiently transfected with c-ret cDNA or with empty plasmidby electroporation (Bio Rad) with −30% efficiency by fluorescence ofcotransfected Red Shift Green Fluorescent Protein in PEF-BOS vector. 48hours later, 10×10⁶ transfected COS cells or 3−5×10⁶ parental COS cellsor NB2/a cells were treated With ¹²⁵I-GDNF, cross linked and analysed asspecified in legends of FIG. 15 and FIG. 16.

[0167] First, the expression of c-RET protein was examined by Westernblotting. c-ret-transfected COS cells (FIG. 18a) and NB2/a cells (notshown) expressed detectable amounts of the c-RET protein, whereas noc-RET protein was detected in mock-transfected (with PBK-CMV plasmid)COS cells (FIG. 18a). PC12 cells also express c-RET protein, albeit atconsiderably lower level than NB2/a cells or c-ret-transfected COS cells(not shown). COS cells, transiently expressing mouse c-retproto-oncogene were incubated with ¹²⁵I-GDNF. As shown in FIG. 17b,those cells bound GDNF, and binding of ¹²⁵I-GDNF can be competed withexcess of unlabeled GDNF. In contrast, no significant binding-of GDNFwas observed in mock-transfected COS cells.

Example 16 Phosphorylation of Tyrosine Residues

[0168] 10×10⁶ transfected COS cells (48 hr after transfection) weretreated with 50 ng/ml of GDNF (Preprotech EC Ldt.) for 5 minutes inserum-free DMEM, or not treated, and then quickly washed with the samemedium. NB2/a cells were similarly treated (results not shown). c-RETproteins were immunoprecipitated from detergent extracts by cocktail ofmonoclonal (Lo, L. & Anderson, D. J. Neuron, 15, 527-539 (1995) andpolyclonal (Santa Cruz) anti-c-ret antibodies, separated by 7% SDS-PAGE,transferred to nitrocellulose, probed by anti-c-ret antibodies (SantaCruz), stripped and reprobed by anti-phosphotyrosine antibodies (Sigma).

[0169] This treatment resulted in significant increase in tyrosinephosphorylation of 190 kD cRET proto-oncogene, the 170 kD form beingless prominently phosphorylated (FIG. 18b). In both cell lines,relatively high c-RET phosphorylation was detected also in the absenceof GDNF (FIG. 18), most probably via endogenous GDNF secreted by thesecells and/or ligand-independent receptor dimerization.

Example 17 ¹²⁵I-GDNF Binds to c-ret-Positive Enteric Neurons

[0170]¹²⁵I-GDNF was bound to developing rat tissue explants in situ. Insitu binding of human ¹²⁵I-GDNF (PeproTech. EC Ltd.), iodinated byChloramine T Method, was carried out essentially as described (Partanenand Thesleff, 1987). Briefly, explants of E15 rat gut were incubatedwith 10 ng/ml of ¹²⁵I-GDNF in Eagle's minimal essential medium on theNuclepore filter (Costar) for 90 min at room temperature. 250-foldexcess of unlabeled GDNF was applied as a competitor to controlexplants. After careful washing, the explants were fixed with 3.5%paraformaldehyde in PBS, sectioned and exposed to NTB-2 emulsion(Kodak).

[0171] The gastrointestinal tract was chosen as it strongly expressesGDNF mRNA (Suvanto et al., 1996); FIGS. 19a and b) and c-RET-positiveneurons are absent in the gastrointestinal tract in c-ret-deficient mice(Schuchart et al., 1994; Durbec et al., 1996). ¹²⁵I-GDNF binds to agroup of cells within the muscle layer of embryonic day (E)15 rat gut(FIGS. 19c and d). This binding was specific as it was totally competedwith 250 fold excess of unlabeled GDNF (FIG. 19h). The cells that bindGDNF were the enteric neurons of the myenteric plexus, as revealed byperipherin immunoreactivity (FIG. 19f).

[0172] Moreover, these neurons also expressed c-ret mRNA, asdemonstrated by in situ hybridization (FIG. 19e). Cloning of the GDNFcDNA and in situ hybridization with GDNF probe was performed exactly asdescribed (Suvanto et al., 1996). A 646 bp long fragment of mouse c-retcDNA (Pachnis et al., 1993) covering the 3′-region of the shorter form(Takahashi et al., 1988) of c-ret was cloned into NotI-Xol site of pBSK+vector (Stratagene). cRNAs in antisense and sense orientation werelabeled with digoxigenin-UTP (Boehringer-Mannheim), hybridized tocryosections through E15 rat gut and visualized with alkalinephosphatase-conjugated anti-goxigenin antibodies according tomanufacturers instructions. In both cases, only background labeling wasobtained with hybridization of corresponding probes in sense orientation(FIG. 19g). Polyclonal anti-peripherin antibodies (Bio-Rad) were appliedto cryosections of E15 rat gut at a dilution of 1:100 for 1 hr andvisualized by FITC-conjugated secondary antibodies (Jackson). Thus, GDNFspecifically binds to c-RET-expressing enteric neurons of developingrat.

Example 18 Affinity-Cross Linking of GDNF to c-RET

[0173] PC12 cells and NB2/a cells were washed three times withserum-free RPMI-1640 or DMEM, respectively, plated on uncoated (NB2/acells) or collagen-coated (PC12 cells) dishes (5000-6000 cells per dishin triplicate) in the presence or absence of 50 ng/ml of GDNF (PeproTechEC Ltd.), and the number of cells microscopically counted after 48 h.

[0174] For c-RET expression in transfected cells, the shorter form(Takahashi el al., 1988) of human wild-type c-ret cDNA was subcloned inpcDNA3 (Invitrogen). 3T3 fibroblasts were stably transfected with c-retexpression plasmid or with empty vector (mock-transfected cells) andpositive cells lines selected with G418.

[0175] Transient transfection of trkC 3T3 fibroblasts (Ip et al. (1993)Neuron, 10,137-149) with human c-ret cDNA in pcDNA3 vector or with emptyvector was performed by the lipofectin method (Gibco-BRI.). c-ret andmock-transfected cells (10.000-15.000 cells per well) in five parallelswere treated with rat GDNF (Trupp et al. (1995) J. Cell. Biol. 130,137-148) at indicated concentrations for five days. NT-3 was used aspositive control at 30 ng/ml. Cell number was quantified by measurementof acid phosphatase activity using Abacus™ Cell Proliferation Kit(Clontech).

[0176] 3−5×10⁶ PC12 cells, NB2/a cells, COS cells or c-ret-3T3 as wellas mock-3T3 cells or mechanically dissociated cells from two F20 or from17 E15 rat kidneys were incubated with 10 ng/ml of ¹²⁵I-GDNF (human GDNFfrom PeproTech EC Ltd. or rat GDNF from C. F. Ibanez) (Trupp et al.,1995), iodinated by Chloramine T method, for 1 hour on ice. 250-foldexcess of unlabeled GDNF (PeproTech EC) or TGF-β1 (kindly provided byDr. M. Laiho) was applied to control sample. ¹²⁵I-GDNF was thencrosslinked to the cells with 30 mM of ethyl-dimethylaminopropylcarbodiimide (EDAC) (Pierce) for 30 minutes on ice. Detergent lysates ofthe cells were immunoprecipitated with polyclonal anti-GDNF antibodies(Santa Cruz) or with the cocktail of monoclonal (kindly provided by Dr.D. Anderson, Lo and Anderson, 1995) and polyclonal (Santa Cruz)anti-c-RET antibodies to neurofilament proteins (a gift of Dr. I.Virtanen) were used as control antibodies. The precipitates werecollected by Protein A-Sepharose (Pharmacia) or by WGA-agarose (a giftfrom Dr. O Renkonen), separated on 7% SDS-PACE, and visualized with aPhosphorimager SI (Molecular DynanLics).

[0177] First, ¹²⁵I-GDNF was cross linked to PC12 cells, NB2/a cells andCOS cells with ethyl-dimethyliaminopropyl carbodiimide (EDAC), and thecomplexes were precipitated with anti-GDNF antibodies. As shown on FIG.20a, complexes with molecular weight of 190 kD and 170 kD were obtainedfrom PC12 and NB2/A cells, but not from COS cells. The molecular weightsof the cross linked proteins (minus GDNF monomer of ˜25K) correspond tothose of c-RET, (140 kD and 160 kD, representing partially and fullyglycosylated isoforms of c-RET, respectively) (Takahashi et al., 1988).

[0178] Next, ¹²⁵I-GDNF was cross linked to PC12 and NB2/a cells by EDACand immunoprecipitated formed complexes with anti-c-RET antibodies. Thebands with molecular weight of 190 kD were obtained from both cell lines(FIGS. 20a and b). Formation of the complexes was abolished by 500-foldexcess of unlabeled GDNF. The reason why both fully and partiallyglycosylated forms of c-RET were precipitated by anti-GDNF antibodies,but only the larger isoform by anti-c-RET antibodies, is unclear. Thesame complexes, although much weaker, were also obtained whendithiobis(succinimidylpropionate) was used as a crosslinker (data notshown).

[0179] The EDAC-crosslink approach was also used to reveal GDNF-c-RETcomplexes from E15 embryonic kidney cells, where c-ret mRNA is stronglyexpressed in the tips of growing ureter branches. With both anti-GDNFand anti-c-RET antibodies, a band of 190 kD was obtained (FIGS. 20a andb) that was competed with excess of unlabeled GDNF. Thus, only the fullyglycosylated form of c-RET is expressed in embryonic kidney cells.

[0180] Cross linked ¹²⁵I-GDNF-c-RET complexes from the cells ectopicallyexpressing c-ret were also demonstrated. 3T3 cells were transfected withc-ret cDNA or with empty plasmid, and established stable transfectedcell lines (c-ret-3T3 cells or mock-3T3 cells). Cross linking of¹²⁵I-GDNF to these cells followed by anti-RET-precipitation revealed a190 kD band that was abolished with 250-fold excess of unlabeled GDNF(FIG. 20b). As GDNF is a distant member of TGF-β family, we also used a250-fold excess of TGF-β1 as a competitor. No competition was observedwith TGF-β1 (FIG. 20b) Taken together, these data show that GDNFdirectly and specifically binds to c-RET.

Example 19 GDNF Specifically Increases Tyrosine Phosphorylation of c-RET

[0181] c-ret-3T3 cells and mock-3T3 cells were treated with GDNF and theproteins from these cells were immunoprecipitated with anti-c-RETantibodies. The precipitated proteins were then analyzed by Westernblotting with anti-phosphotyrosine antibodies.

[0182] 10×10⁶ c-ret-3T3 cells were treated with different doses of GDNF(PeproTech LC Ltd. or from C. F. Ibanez) (Trupp et al., 1995) for 5 min,or with 50 ng/ml of GDNF for indicated times in serum-free Dulbecco'smodified Eagle's medium containing 1 mM Na₃VO₄ and then quickly washedwith the same medium. c-RET proteins were immunoprecipitated fromdetergent extracts, containing 1 mM Na₃VO₄ by cocktail of monoclonal(Lo, L. and Anderson, D. J. (1995) Neuron, 15 527-539) and polyclonal(Santa Cruz) anti-c-RET antibodies, separated by 7% SDS-PAGE andtransfected to nitrocellulose which were probed by anti-phosphotyrosineantibodies to nitrocellulose with was probed by anti-phosphotyrosineantibodies PY20 (Transduction Laboratories), then stripped and reprobedby anti-c-RET antibodies (Santa Cruz).

[0183] As shown on FIG. 21a, a short treatment of c-ret-3T3 cells withGDNF dose-dependently (beginning at 25 ng/ml) increased tyrosinephosphorylation of the 160 kD c-RET isoform, whereas the phosphorylationof the 140 kD isoform remained unchanged. An increase in c-RETphosphorylation was evident at 25 ng/ml of GDNF and above it. No c-RETproteins were detected in mock-3T3 cells. c-ret-3T3 cells were alsotreated with GDNF (50 ng/ml) for different times. An increase in c-RETtyrosine phosphorylation was evident after 5 minutes of treatment andcontinued at least for one hour (FIG. 21b). With prolonged exposition,the increase in phosphorylation of lower c-RET isoform also becameevident. A basal level of c-RET phosphorylation was detected in theabsence of GDNF, possibly via a ligand-independent dimerization of thatreceptor. To reveal the amounts of c-RET protein in these experiments,the filters were stripped from antibodies and reprobed with anti-c-RETantibodies. The level of c-RET protein was not changed by GDNF treatmentin c-ret-3T3 cells (FIGS. 21a and b, lower panels). The finding thatGDNF specifically activates c-RET indicates that c-RET is a signalingreceptor for GDNF.

Example 20 c-RET Expression Confers GDNF-Responsiveness to 3T3 Cells

[0184] Mouse 3T3 fibroblast cell line expressing trkC (trkC-3T3) (Ip etal, 1993) were transiently transformed with c-ret expression plasmid.trkC-3T3 cells die within 2-3 days in serum-free medium in the absenceof trkC ligand neurotrophin-3 (NT-3) (Ip et al., 1993) and do notexpress detectable amounts of c-ret. GDNF dose-dependently increased thenumber of c-ret-transfected but not of mock-transfected trkC-3T3 cells(FIG. 22), which was comparable to the response elicited by NT-3.Whether this is a proliferative or survival-promoting response could notbe distinguished based upon the data. Thus, introduction of c-RET toGDNF-nonresponsive cells is sufficient to bring about the biologicalresponse to GDNF.

Example 21 Isolation of GDNF Receptor

[0185] L6 myoblast cells were lysed with 1% NP40 and cell lysates werefractionated by anionic exchange on a Q-Sepharose column. Fractionseluted at different ionic strength were dialyzed and assayed for bindingto GDNF immoliblized on a chip in a Biacore device (Pharmacia). Adistinct binding component was detected in a fraction of total proteinmixture. The equivalent fraction of a COS cell lysate did not showbinding under the same conditions (data not shown).

[0186] Further purification of the GDNF binding activity in the first 1Msalt fraction was obtained after hydrophobic interaction chromatography(FIG. 23b). The data represents the ratio between GDNF binding (inresonance units) and protein concentration (OD at 280 nm). Fractionswere eluted with a step-wise gradient of ammonium sulfate.

[0187] Alternatively, purification may be effected by cross linking GDNFto cells in the presence of tracer amounts of radiolabeled ligand, andligand/receptor complexes can by fractionated through ion exchangechromatography followed by hydrophobic interaction chromatography andSDS/PAGE. Bands corresponding to the molecular weights of GDNF-receptorcomplexes can by excised, dissociated, and then sequenced by massspectrometry or Edman degradation, depending upon the yield of recovery.

Example 22 A Novel GDNF-Binding Protein in Brain

[0188] By ligand blotting, we have identified another GDNF-bindingprotein from total brain extract. We bound ¹²⁵I-GDNF to the filterscarrying protein from the total extracts of brain and liver (a ligandblot assay). A major band with MW of about 50 kD was obtained from brainextract, but not from liver (FIG. 24). This binding is specific as¹²⁵I-GDNF did not bind to other proteins from total lysates, it is notfound in liver lysates (nor in some other tissues), and it can becompeted with excess of unlabeled GDNF.

[0189] Binding of ¹²⁵I-GDNF to c-RET was not revealed in the ligandblots. The reason for this may be the very low share of c-RET in totalbrain extract. Alternatively, by analogy with other receptors for GDNF,c-RET might not bind GDNF directly, but might first bind to anothernonsignalling receptor that thereafter presents the ligand to c-RET, asignaling receptor. A 50 kD GDNF-binding protein is a good candidate forthe putative presenting receptor.

Example 23 Protocol for Isolating Novel Signaling Receptors for GDNF

[0190] In the absence of serum, 3T3 fibroblasts can be made dependent ona given exogenous growth factor provided appropriate receptors areexpressed on the cell surface. An expression library can be made usingRN33B cDNA, which can then be transfected into 3T3 fibroblasts byprocedures well known in the art (Maniatis et al., supra). Stabletransfectants can be selected in serum-free media supplemented withGDNF. Fibroblast clones that express signaling GDNF receptors willselectively grow in the presence of GDNF in serum-free media. Theselection step may allow detection of even very reare clones due totheir differential growth advantage. Further analysis of the recoveredclones in media with or without GDNF would help to distinguishGDNF-dependent from GDNF-independent survival of clones.

Example 24 Cloning and Sequencing of GDNFR-β Rat

[0191] A BLAST search of the publicly available genomic database at NCBI(National Center for Biotechnology Information) was performed using theamino acid sequence for GDNFR-α as reported in Jing et al. (1996) Cell85:1113-1124 (hereby incorporated by reference). The search revealedseveral human ESTs with homology to the GDNFR-α. Primers were designedfrom these sequences and a partial cDNA clone was amplified from humanfetal brain RNA. The upstream PCR primer was5′ATGGATCCGCAACCTGAATGACAACTGC3′ [SEQ ID NO: 3]. The downstream PCRprimer was 5′CCGAATTCAGTTGGGCTTCTCCTlTGTC3′ [SEQ ID NO: 4]. This cDNAclone was used to screen a rat brain cDNA library from which a nearlycomplete cDNA was isolated. Additional PCRs were used to clone the most5′ of this cDNA. The complete cDNA sequence is depicted in FIG. 32 (SEQID NO:5). The amino acid sequence of the complete cDNA clone wasdetermined and is depicted in FIG. 25. It is 464 amino acids long. Aputative signal peptide and a GPI anchoring motif were identified in theN-terminal 15 and C-terminal 17 amino acid residues, respectively.

Human

[0192] Rat GDNFR-α sequence (6 GenBank™ accession number U59486) wasused to screen the human EST database and 8 human sequences from 6different clones (GenBank™ accession numbers H12981, H05619, R02135,R02249, T03342, W73681, W73633 and Z43761) with high similarity(identity >70% over 200 bp) were identified. Full sequence analysis ofthree EST clones revealed a 3′-terminal consensus sequence which wedesignated human EST GDNFR-β cDNA (nucleotides 469-1490 in FIG. 33).

[0193] To obtain complete cDNA, human EST GDNFR-β cDNA was used forscreening an adult rat hippocampus cDNA library (λZap, Stratagene). Twoidentical clones out of one million contained a 2002 base pair longsequence with 91% identity to the probe. With a forward primer designedfrom rat GDNFR-β sequence, the 5′ end of human GDNFR-β was amplified byPCR from human total brain cDNA. Human total RNA was extracted bystandard methods and reverse transcribed in a random-primed reaction asdescribed in Superscript II (Life Technologies) protocol. The humanGDNFR-β gene was amplified from cDNA under the following PCR conditions:dNTP's in 200 μM concentration and primers (forward)5′-ATGATCTTGGCAAACGCCTTCTG-3 [SEQ ID NO: 6] and (reverse)5′-TTGCAGTTGTCATTCAGGTTGC-3′ [SEQ ID NO: 7] in 1 μM concentration,approximately 5 ng of human brain cDNA, 1 u of Dynazyme (Finnzymes) Taqpolymerase in 50 μl. The 30 cycles after initial 5 minutes at 94° C.consisted of 30 s at 94°, 30 s at 57° C. and 1 min at 72° C. with afinal 5 minute extension at 72° C. The PCR fragments were cloned intoPGEM-T vector (Promega) and four different clones were sequenced. Withseveral primer pairs complete GDNFR-β cDNA sequence was amplified by PCRfrom the same human brain cDNA. This sequence was identical to theEST-derived sequence. The overlapping inserts of EST and PCR fragmentswere combined and cloned to get the contig of the full-length humancDNA. The human GDNFR-β cDNA sequence (GenBank I accession numberU93703) contains a 1395 base pair long open reading frame (FIG. 33).

[0194] At amino acid level, human and rat GDNFR-β orthologues were 96%identical. The predicted 47 kD (unglycosylated) mature protein, consistsof 464 amino acids that are 48% identical and up to 63% similar to thepublished sequence of human and rat GDNFR-α proteins (Jing et al.,supra) (FIG. 26). Alignment was performed using MAP (multiple alignmentprogram). (Huang, X, Comp. Appl. BioSci., 10: 227-235, 1994. Theputative signal sequence was predicted according to von Heijne, Nucl.Acids. Res., 14:4683-4699, 1986). The amino acid sequence of GDNFR-β hasa putative signal sequence, three N-glycosylation sites, and a putativeGPI-anchor site similar to GDNFR-α. Completely conserved cysteinresidues and strong overall resemblance to GDNFR-α predict highsimilarity in the spatial structures of the GDNF-receptors α and β (FIG.26).

[0195] Amino acid identity as used herein means the same amino acidresidue. Amino acid similarity as used herein means the same orchemically similar amino acid residue (e.g., Lys is identical to Lys,but is similar to Arg).

Example 25 GDNFR-α and GDNFR-β Expression

[0196] Expression of mRNA for GDNFR-α and GDNFR-β was investigated byRNAse protection analysis, as disclosed in Example 4 above, in cellsknown to express GDNF receptors as determined from chemical crosslinking studies. GDNFR-c mRNA was found in RN33B and MN1 cells (FIG.27). High levels of GDNFR-β mRNA were detected in RN33B cells. Lowerlevels were found in post-natal (P1) kidney and in MN1 cells. (See FIG.28.) No GDNFR-α or GDNFR-β mRNA could be detected in L6 cells (FIGS. 27and 28).

[0197] The mRNA expression patterns of GDNFR-α and GDNFR-β mRNAs werealso determined and compared in E17 rat embryo by in situ hybridization.In situ hybridization on E17 rat sections was performed exactly asdescribed previously (Suranto et al. Eur. J. Neurosc., 8:816-822, 1996;Wilkinson et al. in Post Implantation Mammalian Embryos a PracticalApproach, IRL Press, Oxford, Copp., A. J. and Cockroft, D. L., eds. pp.151-171, 1990, both incorporated herein by reference). The antisensecRNA probes for rat GDNFR-α and rat GDNFR-β covered nucleotides 294-1039of GenBank™ sequence U59486 and nucleotides 1231-1394 of the rat GDNFR-βsequence, respectively. In several organs, GDNFR-α and GDNFR-β mRNAsshowed distinct, non-overlapping distributions. Strong GDNFR-β mRNAexpression was seen in the capsule and cortex of adrenal gland (FIG.37B), whereas GDNFR-α mRNA was detected in adrenal medulla (FIG. 37C).In kidney, GDNFR-α transcripts were seen in the tips of ureter bud,condensed mesenchyme and early epithelial tubules (FIG. 37F), whereasGDNFR-β mRNA was present in undifferentiated nephrogenic mesenchyme andin the muscle wall of renal pelvis (FIG. 37E). Strong expression ofGDNFR-α mRNA was present in the muscle and nervous layers alonggastrointestinal tract (FIG. 37I). In stomach, GDNFR-β mRNA wasmoderately expressed in nervous layers (not shown), while in entericplexus of small intestine only low amounts of transcripts were detected(FIG. 37H). In embryonic (E)17 rat nervous system, GDNFR-α mRNA wasabundantly expressed in spinal cord, especially in ventral motoneurons(FIG. 37J). Also GDNFR-β mRNA was present in many areas of spinal cordincluding ventral motoneurons (FIGS. 5J, K), though the levels weremoderate. In dorsal root and trigeminal ganglia, GDNFR-β mRNA expressionwas restricted to subpopulations of neuronal cells (FIGS. 37K,M,N),resembling Ret mRNA distribution (Pachnis et al., Development,119:1005-1017, 1993). In contrast, varying amounts of GDNFR-αtranscripts could be detected throughout the ganglia (FIGS. 37J,L,M,O)and also in the cells covering trigeminal and spinal nerves (FIGS. 37M,O), suggesting expression in both neuronal and glial cells.

Example 26 Affinity Cross Linking of GDNF to GDNFR-α and GDNFR-β

[0198] COS cells were transfected with GDNFR-α, GDNFR-β, c-RET, and acontrol plasmid and affinity cross lining studies were performed asdisclosed in Example 15 above. The results are depicted in FIG. 29. COScells transfected with a control plasmid did not bind GDNF (lanes 1 and2). Transfection with a GDNFR-α cDNA followed by incubation with¹²⁵I-GDNF generated a GDNF binding complex with an approximate molecularweight of 70 kD (lane 3). The formation of this complex could beprevented by incubation with cold GDNF (lane 4).

[0199] Transfection with a GDNFR-β cDNA generated a GDNF binding complexwith an approximate molecular weight of 100 kD (lane 5). The formationof this complex could also be prevented by incubation with cold GDNF(lane 6). Both GDNFR-α and GDNFR-β facilitated specific cross linking of¹²⁵I-GDNF to c-RET (lanes 7 to 10). COS cells transfected with a c-RetcDNA alone did not bind GDNF (lanes 11 and 12). Molecular weight markersand the positions of the different GDNF receptor complexes areindicated.

Example 27 Comparison with Complexes from Cross Linking Studies withRN33B Cells

[0200] When compared side-by-side, the complex of GDNFR-α and GDNFformed in the transfected COS cells ran at the same molecular weight asthe 70 kD complex detected in RN33B cells after GDNF cross linking withEDAC performed as disclosed in Example 2 (FIG. 30, compare lanes 1 and2). The complex of GDNFR-β and GDNF formed in the transfected COS cellsran at the same molecular weight as the 100 kD complex detected in RN33Bcells (compare lanes 1 and 3). The identity of the receptor component inthe 155 kD binding complex is unknown, but it is formed by binding tothe 135 k GDNF-binding protein identified above.

Example 28 Phosphorylation of cRET

[0201] Upon transfection into COS cells, cRET is highly phosphorylatedon tyrosine residues independent of the presence or absence of GDNF(FIG. 31, upper panel). COS cells were transfected with 4 μg of c-Retplasmid and 16 μg control plasmid. Transfected COS cells were exposed todifferent concentrations of GDNF using the procedure disclosed inExample 16, and cytoplasmic cell lysates were immunoprecipitated withanti-c-RET antibodies. Precipitated c-RET receptors were analyzed fortyrosine phosphorylation by Western blotting using anti-P-tyrosinemonoclonal antibodies.

[0202] Co-transfection with plasmids encoding GDNFR-α or GDNFR-β (inplace of the control plasmid), although allowing for ligand stimulationof receptor tyrosine phosphorylation in a dose-dependent manner,resulted in a dramatic attenuation of the steady-state levels oftyrosine phosphorylation of c-RET, i.e., in the absence of GDNF (FIG.31, upper panel). Reprobing of the same blot with anti-c-RET antibodiesrevealed that all lanes contained equal amounts of c-RET (data notshown).

[0203] When COS-7 cells were transiently transfected with cDNAs encodingGDNF receptors in different combinations and treated with GDNF (100ng/ml), it induced phosphorylation of the 170 kD formm of Ret only inthe presence of either GDNFR-α or GDNFR-β (FIG. 36, lanes 14), but notwithout these receptors (FIG. 36, lanes 5-6). The 170 kD from of Retrepresents the fully glycosylated mature receptor on the plasma membrane(Takahashi et al. Oncogene, 3:571-578, 1988). In control cellscotransfected with GDNFR-α and GDNFR-β but without Ret, no tyrosinephosphorylated proteins were precipitated by anti-Ret antibodies (FIG.36, lanes 7-8). Identical results were gained when a GDNFR-β constructlacking GPI-anchor was used (data not shown). Tyrosine phoshorylation ofimmature, partially glycosylated 150 kD form of Ret was not increased byGDNF treatment. The nature of the 85 kD bands in GDNFR-α transfectedcells is not known. The major 50 kD band in all lanes is the heavy chainof IgG.

[0204] Human full-length GDNFR-β cDNA was cloned into pCDNA3(Invitrogen) and pBk-CMV (Stratagene) mammalian expression vectors. RatGDNF-β cDNA was cloned into the same expression vectors. In one ratconstruct, the 3′ end of human GDNFR-β cDNA was added using a uniqueBclI restriction site and in another construct, an artificial stop codonwas inserted instead of GPI-tail, producing an apparent soluble form ofrat GDNFR-β.

[0205] COS-7 cells (5×10⁶ cells per experimental point) werecotransfected by electroporation (Bio-Rad) with cDNAs (5 μg each) of Retand GDNFR-α, Ret and GDNFR-β, GDNFR-α, and GDNFR-β, or with cDNA of Retalone and cultured for 48 hours. Cellular phosphatases were inhibited by1 mM Na₃, VO⁴ for one hour, the cells were treated with 100 ng/ml ofGDNF (Promega or PeproTech Ltd) for 30 minutes and lysed inTris-balanced saline. pH 7.5, containing 2 mM EDTA, 10% glycerol, 1%Nonidet P-40, 1% Triton x-100 and protease inhibitors. Proteinsimmunoprecipitated by anit-RET antibodies (Santa Cruz) were analysed byWestern blotting with anti-phosphotyrosine antibodies PY20 (TransductionLaboratories). In experiments with the secreted form of GDNF-β lackingan GPI anchor, the cells were not washed before GDNF treatment.

[0206] Neuro-2A cells express c-RET endogenously, but show low levels ofconstitutive receptor tyrosine phosphorylation (FIG. 31, lower panel).However, transfection of the Neuro-2A cells using procedures disclosedabove resulted in stimulation of tyrosine phosphorylation of the c-RETreceptor in the presence of GDNF in a dose-dependent manner (FIG. 31,lower panel).

Example 29 Tissue Distribution of GDNFR-β

[0207] The tissue distribution of human GDNFR-β was studied by Northernhybridisation of mRNA extracted from different adult and fetal tissues.The expression of GDNFR-β mRNA was abundant in adult brain, intestineand placenta, as well as in fetal brain, lung and kidney. Two majortranscripts of 2.9 and 3.5 kb were visible in all tissues, andadditional transcripts of 7.5 kb in placenta and 1.4 kb in testis (FIG.34) were found.

[0208] For Northern Hybridization, 100 ng of the human EST GDNFR-βinsert was labeled with ³²P-DCTP (Amersham) by Prime-a-Gene-Kit(Promega). The specific activity of the final probe was 2×10⁷ cpm/μg andthe hybridization of Human and Human Fetal Multiple Tissue Northern Blotfilters (Clontech) was performed in ExpressHyb solution at 65° for 2hours. The filters were washed two times for 30 minutes at 50° C. in 2×saline sodium citrate (SSC) +0.1% SDS and 0.1% SSC+0.1% SDS and thenanalysed by phosphoimager (Fuji BAS 1500). As a control, the samefilters were hybridized with human β-actin probe (Clontech).

Example 30 GDNFR-β Locus

[0209] The chromosomal locus of GDNFR-β was assigned to human chromosome8p21-22 by fluorescent in situ hybridization (FISH) with the 1.49 kbhuman GDNFR-β cDNA probe. In addition, the locus for mouse GDNFR-β genewas assigned to the mouse chromosome 14D3-E1 that corresponds to thehuman locus 8p21-22 (FIG. 35).

[0210] Human peripheral blood lymphocytes were cultured and the cellculture from a mouse fetal tissue was established according to standardprotocols (Fresney, I. R., Culture of Animal Cells, Manual of BasicTechniques, Allan R. Lisa, Inc. New York, 1983) and used as a source ofmetaphases chromosomes. Both human lymphocytes and mouse monolayer cellswere treated with 5-bromeoxyuridine at early replicating phase to inducea banding pattern (Takanashi, et al. Hum. Gene, 86:14-16, 1990; Lemieuxet al., Cell Gene, 59:311-312, 1992). The slides were stained withHoechst 33258 (1 μg/ml) and exposed to UV-light. (302 nm) for 30 min.The probes were labeled by a nick translation kit (BRL) withbiotin-11-dUTP (Sigma) and the FISH procedure was carried out in 50%formamide, 10% dextran sulphate in 2×SSC as described earlier (Lichteret al., Proc. Natl. Acad. Sci. USA, 85:9664-9665, 1988; Pinkel et al,Proc. Natl. Acad. Sci., USA, 83:2934-2938, 1986, both incorporatedherein by reference). Repetitive sequences were suppressed with 10-foldexcess of Cot-1-DNA (BRL) and after overnight incubation unspecifichybridization signals were eliminated by washing the slides with 50%formamide/ 2×SSC, 2×SSC and 0.5×SSC at 45° C. Specific hybridizationsignals were visualized using FITC-conjugated avidin (VecotrLaboratories) and slides were counterstained with4′-6′-diamino-2-phenylindole (25 ng/ml). The image analysis foracquisition, display and quantification of hybridization signals wasperformed with a PXL camera (Photometrics) attached to a PowerMac7100/AV workstation. IPLab software controls the camera operation, imageacquisition and Ludl wheel (Heiskanen, et al. Genet. Anal. Biol. Eng.,12:179-184, 1996). The probe for human GDNFR-β gene was 1490 bp longcDNA and the hybridization showed specific double spot signal in 30 outof 100 metaphase chromosomes that were identified based on theirG-banding pattern. The hybridization signal of the 10 kb genomic mouseprobe showed specific localization in 27 out of 30 mouse metaphasechromosomes (Cowell, et al., Chromosome, 89:294320, 1984).

[0211] All references cited herein are hereby incorporated by referencein their entireties.

1 11 1 468 PRT Rattus sp. 1 Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu ProLeu Leu Asp Leu Leu 1 5 10 15 Met Ser Ala Glu Val Ser Gly Gly Asp ArgLeu Asp Cys Val Lys Ala 20 25 30 Ser Asp Gln Cys Leu Lys Glu Gln Ser CysSer Thr Lys Tyr Arg Thr 35 40 45 Leu Arg Gln Cys Val Ala Gly Lys Glu ThrAsn Phe Ser Leu Thr Ser 50 55 60 Gly Leu Glu Ala Lys Asp Glu Cys Arg SerAla Met Glu Ala Leu Lys 65 70 75 80 Gln Lys Ser Leu Tyr Asn Cys Arg CysLys Arg Gly Met Lys Lys Glu 85 90 95 Lys Asn Cys Leu Arg Ile Tyr Trp SerMet Tyr Gln Ser Leu Gln Gly 100 105 110 Asn Asp Leu Leu Glu Asp Ser ProTyr Glu Pro Val Asn Ser Arg Leu 115 120 125 Ser Asp Ile Phe Arg Ala ValPro Phe Ile Ser Asp Val Phe Gln Gln 130 135 140 Val Glu His Ile Ser LysGly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145 150 155 160 Cys Asn Leu AspAsp Thr Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr 165 170 175 Pro Cys ThrThr Ser Met Ser Asn Glu Val Cys Asn Arg Arg Lys Cys 180 185 190 His LysAla Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser 195 200 205 TyrGly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg 210 215 220Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Arg 225 230235 240 Pro Asn Cys Leu Ser Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys245 250 255 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu SerArg 260 265 270 Ser Val Ser Asn Cys Leu Lys Glu Asn Tyr Ala Asp Cys LeuLeu Ala 275 280 285 Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn TyrVal Asp Ser 290 295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys SerAsn Ser Gly Asn 305 310 315 320 Asp Leu Glu Asp Cys Leu Lys Phe Leu AsnPhe Phe Lys Asp Asn Thr 325 330 335 Cys Leu Lys Asn Ala Ile Gln Ala PheGly Asn Gly Ser Asp Val Thr 340 345 350 Met Trp Gln Pro Ala Pro Pro ValGln Thr Thr Thr Ala Thr Thr Thr 355 360 365 Thr Ala Phe Arg Val Lys AsnLys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 Asn Glu Ile Pro Thr HisVal Leu Pro Pro Cys Ala Asn Leu Gln Ala 385 390 395 400 Gln Lys Leu LysSer Asn Val Ser Gly Ser Thr His Leu Cys Leu Ser 405 410 415 Asp Ser AspPhe Gly Lys Asp Gly Leu Ala Gly Ala Ser Ser His Ile 420 425 430 Thr ThrLys Ser Met Ala Ala Pro Pro Ser Cys Ser Leu Ser Ser Leu 435 440 445 ProVal Leu Met Leu Thr Ala Leu Ala Ala Leu Leu Ser Val Ser Leu 450 455 460Ala Glu Thr Ser 465 2 464 PRT Rattus sp. 2 Met Ile Leu Ala Asn Ala PheCys Leu Phe Phe Phe Leu Asp Glu Thr 1 5 10 15 Leu Arg Ser Leu Ala SerPro Ser Ser Leu Gln Gly Ser Glu Leu His 20 25 30 Gly Trp Arg Pro Gln ValAsp Cys Val Arg Ala Asn Glu Leu Cys Ala 35 40 45 Ala Glu Ser Asn Cys SerSer Arg Tyr Arg Thr Leu Arg Gln Cys Leu 50 55 60 Ala Gly Arg Asp Arg AsnThr Met Leu Ala Asn Lys Glu Cys Gln Ala 65 70 75 80 Ala Leu Glu Val LeuGln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 85 90 95 Arg Gly Met Lys LysGlu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile 100 105 110 His Leu Gly LeuThr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr 115 120 125 Glu Pro ValThr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile 130 135 140 Phe SerGly Thr Gly Thr Asp Pro Ala Val Ser Thr Lys Ser Asn His 145 150 155 160Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys 165 170175 Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr 180185 190 Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe195 200 205 Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys SerCys 210 215 220 Gln Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile LeuPro Ser 225 230 235 240 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys LeuAsp Leu Arg Ser 245 250 255 Leu Cys Arg Thr Asp His Leu Cys Arg Ser ArgLeu Ala Asp Phe His 260 265 270 Ala Asn Cys Arg Ala Ser Tyr Arg Thr IleThr Ser Cys Pro Ala Asp 275 280 285 Asn Tyr Gln Ala Cys Leu Gly Ser TyrAla Gly Met Ile Gly Phe Asp 290 295 300 Met Thr Pro Asn Tyr Val Asp SerAsn Pro Thr Gly Ile Val Val Ser 305 310 315 320 Pro Trp Cys Asn Cys ArgGly Ser Gly Asn Met Glu Glu Glu Cys Glu 325 330 335 Lys Phe Leu Arg AspPhe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile 340 345 350 Gln Ala Phe GlyAsn Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro 355 360 365 Ser Leu ProAla Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu 370 375 380 Pro AspAsp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr 385 390 395 400Thr Cys Thr Ser Ile Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys 405 410415 Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly 420425 430 Ser Lys Lys Val Ile Lys Leu Asn Ser Gly Ser Ser Arg Ala Arg Leu435 440 445 Ser Ala Ala Leu Thr Ala Leu Pro Leu Leu Met Leu Thr Leu AlaLeu 450 455 460 3 28 DNA Artificial Sequence Description of ArtificialSequence Primer 3 atggatccgc aacctgaatg acaactgc 28 4 27 DNA ArtificialSequence Description of Artificial Sequence Primer 4 ccgaattcagttgggcttct ccttgtc 27 5 1414 DNA Rattus sp. 5 atgatcttgg caaacgccttctgcctcttc ttctttttag acgaaaccct ccgctctttg 60 gccagccctt cctccctgcagggctctgag ctccacggct ggcgccccca agtggactgt 120 gtccgggcca atgagctgtgtgcggctgaa tccaactgca gctccaggta ccgcaccctt 180 cggcagtgcc tggcaggccgggatcgcaat accatgctgg ccaataagga gtgccaggca 240 gccctggagg tcttgcaggaaagcccactg tatgactgcc gctgcaagcg gggcatgaag 300 aaggagctgc agtgtctgcagatctactgg agcatccatc tggggctgac agagggtgag 360 gagttctatg aagcttccccctatgagcct gtgacctcgc gcctctcgga catcttcagg 420 ctcgttcaat tcttctcagggacagggaca gacccggcag tcagtaccaa aagcaaccac 480 tgcctggatg ccgccaaggcctgcaacctg aatgacaact gcaagaagct tcgctcctct 540 tatatctcca tctgcaaccgtgagatctct tgagatctct cccaccgaac gctgcaaccg 600 cacaaggctc tgcgccagttctttgaccgt gtgcccagcg agtataccta ccgcatgctc 660 ttctgctcct gtcaggaccaggcatgtgct gagcgtcgcc ggcaaaccat cctgcccagt 720 tgctcctatg aggacaaggagaagcccaac tgcctggacc tgcgcagcct gtgtcgtaca 780 gaccacctgt gccggtcccgactggcagat ttccacgcca actgtcgagc ctcctaccgg 840 acaatcacca gtcgtcctgcggacaactac caggcatgtc tgggctccta tgctggcatg 900 attgggtttg atatgacacccaactatgtg gactccaacc ccacgggcat cgtggtgtct 960 ccctggtgca attgtcgtggcagtgggaac atggaagaag agtgtgagaa gttcctcagg 1020 gacttcacgg aaaacccatgcctccggaat gccattcagg cctttggtaa tggcacagat 1080 gtgaacatgt ctcccaaaggcccctcactc ccagctaccc aggcccctcg ggtggagaag 1140 actccttcac tgccagatgacctcagtgac agcaccagcc tggggaccag tgtcatcacc 1200 acctgcacat ctatccaggagcaagggctg aaggccaaca actccaaaga gttaagcatg 1260 tgcttcacag agctcacgacaaacatcagt ccagggagta aaaaggtgat caaacttaac 1320 tcaggctcca gcagagccagactgtcggct gccttgactg ccctcccact cctgatgctg 1380 accttggcct tgtaggcctttggaacccag caca 1414 6 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 6 atgatcttgg caaacgcctt ctg 23 7 22 DNAArtificial Sequence Description of Artificial Sequence Primer 7ttgcagttgt cattcaggtt gc 22 8 465 PRT Homo sapiens 8 Met Phe Leu Ala ThrLeu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 Leu Ser Ala GluVal Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 Ser Asp Gln CysLeu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 Leu Arg Gln CysVal Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 50 55 60 Gly Leu Glu AlaLys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 Gln Lys SerLeu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95 Lys Asn CysLeu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly 100 105 110 Asn AspLeu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125 SerAsp Ile Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln 130 135 140Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145 150155 160 Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr165 170 175 Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg LysCys 180 185 190 His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala LysHis Ser 195 200 205 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala CysThr Glu Arg 210 215 220 Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr GluGlu Arg Glu Lys 225 230 235 240 Pro Asn Cys Leu Asn Leu Gln Asp Ser CysLys Thr Asn Tyr Ile Cys 245 250 255 Arg Ser Arg Leu Ala Asp Phe Phe ThrAsn Cys Gln Pro Glu Ser Arg 260 265 270 Ser Val Ser Ser Cys Leu Lys GluAsn Tyr Ala Asp Cys Leu Leu Ala 275 280 285 Tyr Ser Gly Leu Ile Gly ThrVal Met Thr Pro Asn Tyr Ile Asp Ser 290 295 300 Ser Ser Leu Ser Val AlaPro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 Asp Leu Glu GluCys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 Cys Leu LysAsn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 Val TrpGln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr 355 360 365 ThrAla Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala 385 390395 400 Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser405 410 415 Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His IleThr 420 425 430 Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser ProLeu Leu 435 440 445 Val Arg Val Val Thr Ala Leu Ser Thr Leu Leu Ser LeuThr Glu Thr 450 455 460 Ser 465 9 464 PRT Homo sapiens 9 Met Ile Leu AlaAsn Ala Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr 1 5 10 15 Leu Arg SerLeu Ala Ser Pro Ser Ser Leu Gln Gly Pro Glu Leu His 20 25 30 Gly Trp ArgPro Pro Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala 35 40 45 Ala Glu SerAsn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu 50 55 60 Ala Gly ArgAsp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala 65 70 75 80 Ala LeuGlu Val Leu Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 85 90 95 Arg GlyMet Lys Lys Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile 100 105 110 HisLeu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr 115 120 125Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile 130 135140 Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His 145150 155 160 Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys LysLys 165 170 175 Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Glu Ile SerPro Thr 180 185 190 Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu ArgGln Phe Phe 195 200 205 Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met LeuPhe Cys Ser Cys 210 215 220 Gln Asp Gln Ala Cys Ala Glu Arg Arg Arg GlnThr Ile Leu Pro Ser 225 230 235 240 Cys Ser Tyr Glu Asp Lys Glu Lys ProAsn Cys Leu Asp Leu Arg Gly 245 250 255 Val Cys Arg Thr Asp His Leu CysArg Ser Arg Leu Ala Asp Phe His 260 265 270 Ala Asn Cys Arg Ala Ser TyrGln Thr Val Thr Ser Cys Pro Ala Asp 275 280 285 Asn Tyr Gln Ala Cys LeuGly Ser Tyr Ala Gly Met Ile Gly Phe Asp 290 295 300 Met Thr Pro Asn TyrVal Asp Ser Ser Pro Thr Gly Ile Val Val Ser 305 310 315 320 Pro Trp CysSer Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu 325 330 335 Lys PheLeu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile 340 345 350 GlnAla Phe Gly Asn Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro 355 360 365Ser Phe Gln Ala Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu 370 375380 Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr 385390 395 400 Thr Cys Thr Ser Val Gln Glu Gln Gly Leu Lys Ala Asn Asn SerLys 405 410 415 Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn Ile IlePro Gly 420 425 430 Ser Asn Lys Val Ile Lys Pro Asn Ser Gly Pro Ser ArgAla Arg Pro 435 440 445 Ser Ala Ala Leu Thr Val Leu Ser Val Leu Met LeuLys Gln Ala Leu 450 455 460 10 1490 DNA Homo sapiens 10 atgatcttggcaaacgcctt ctgcctcttc ttctttctag acgagaccct ccgctctttg 60 gccagcccttcctccctgca gggccccgag ctccacggct ggcgcccccc agtggactgt 120 gtccgggccaatgagctgtg tgccgccgaa tccaactgca gctctcgcta ccgcactctg 180 cggcagtgcctggcaggccg cgaccgcaac accatgctgg ccaacaagga gtgccaggcg 240 gccttggaggtcttgcagga gagcccgctg tacgactgcc gctgcaagcg gggcatgaag 300 aaggagctgcagtgtctgca gatctactgg agcatccacc tggggctgac cgagggtgag 360 gagttctacgaagcctcccc ctatgagccg gtgacctccc gcctctcgga catcttcagg 420 cttgcttcaatcttctcagg gacaggggca gacccggtgg tcagcgccaa gagcaaccat 480 tgcctggatgctgccaaggc ctgcaacctg aatgacaact gcaagaagct gcgctcctcc 540 tacatctccatctgcaaccg cgagatctcg cccaccgagc gctgcaaccg ccgcaagtgc 600 cacaaggccctgcgccagtt cttcgaccgg gtgcccagcg agtacaccta ccgcatgctc 660 ttctgctcctgccaagacca ggcgtgcgct gagcgccgcc ggcaaaccat cctgcccagc 720 tgctcctatgaggacaagga gaagcccaac tgcctggacc tgcgtggcgt gtgccggact 780 gaccacctgtgtcggtcccg gctggccgac ttccatgcca attgtcgagc ctcctaccag 840 acggtcaccagctgccctgc ggacaattac caggcgtgtc tgggctctta tgctggcatg 900 attgggtttgacatgacacc taactatgtg gactccagcc ccactggcat cgtggtgtcc 960 ccctggtgcagctgtcgtgg cagcgggaac atggaggagg agtgtgagaa gttcctcagg 1020 gacttcaccgagaacccatg cctccggaac gccatccagg cctttggcaa cggcacggac 1080 gtgaacgtgtccccaaaagg cccctcgttc caggccaccc aggcccctcg ggtggagaag 1140 acgccttctttgccagatga cctcagtgac agtaccagct tggggaccag tgtcatcacc 1200 acctgcacgtctgtccagga gcaggggctg aaggccaaca actccaaaga gttaagcatg 1260 tgcttcacagagctcacgac aaatatcatc ccagggagta acaaggtgat caaacctaac 1320 tcaggccccagcagagccag accgtcggct gccttgaccg tgctgtctgt cctgatgctg 1380 aaacaggccttgtaggctgt gggaaccgag tcagaagatt tttgaaacta cgcagacaag 1440 aacagccgcctgacgaaatg gaaacacaca cagacacaca cacaccttgc 1490 11 445 PRT Rattus sp.11 Met Ile Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr 1 510 15 Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gln Gly Ser Glu Leu His 2025 30 Gly Trp Arg Pro Gln Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala 3540 45 Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu 5055 60 Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala 6570 75 80 Ala Leu Glu Val Leu Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys85 90 95 Arg Gly Met Lys Lys Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile100 105 110 His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser ProTyr 115 120 125 Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu AlaSer Ile 130 135 140 Phe Ser Gly Thr Gly Thr Asp Pro Ala Val Ser Thr LysSer Asn His 145 150 155 160 Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu AsnAsp Asn Cys Lys Lys 165 170 175 Leu Arg Ser Ser Tyr Ile Ser Ile Cys AsnArg Glu Ile Ser Pro Thr 180 185 190 Glu Arg Cys Asn Arg Arg Lys Cys HisLys Ala Leu Arg Gln Phe Phe 195 200 205 Asp Arg Val Pro Ser Glu Tyr ThrTyr Arg Met Leu Phe Cys Ser Cys 210 215 220 Gln Asp Gln Ala Cys Ala GluArg Arg Arg Gln Thr Ile Leu Pro Ser 225 230 235 240 Cys Ser Tyr Glu AspLys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser 245 250 255 Leu Cys Arg ThrAsp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His 260 265 270 Ala Asn CysArg Ala Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp 275 280 285 Asn TyrGln Ala Cys Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp 290 295 300 MetThr Pro Asn Tyr Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser 305 310 315320 Pro Trp Cys Asn Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu 325330 335 Lys Phe Leu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile340 345 350 Gln Ala Phe Gly Asn Gly Thr Asp Val Asn Met Ser Pro Lys GlyPro 355 360 365 Ser Leu Pro Ala Thr Gln Ala Pro Arg Val Glu Lys Thr ProSer Leu 370 375 380 Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr SerVal Ile Thr 385 390 395 400 Thr Cys Thr Ser Ile Gln Glu Gln Gly Leu LysAla Asn Asn Ser Lys 405 410 415 Glu Leu Ser Met Cys Phe Thr Glu Leu ThrThr Asn Ile Ser Pro Gly 420 425 430 Ser Lys Lys Val Ile Lys Leu Asn SerGly Ser Ser Leu 435 440 445

What is claimed is:
 1. An isolated receptor which binds glial celllined-derived neurotrophic factor (GDNF), said receptor comprising atleast one polypeptide having a molecular weight selected from the groupconsisting of polypeptides of about 55 kD, 70 kD, 135 kD, and 300 kDmolecular weight, as determined by SDS-PAGE on 4-20% gradient gels.
 2. Acompetitive assay for identifying compounds which bind to GDNF receptorscomprising a) incubating said compounds with cells which express c-RETreceptors in the presence of an excess of labeled GDNF; b) measuring theamount of labeled GDNF bound to said cells; and c) comparing amountlabeled GDNF bound to said cells to that of controls not incubated withsaid compounds.
 3. The method of claim 2 wherein the cells are selectedfrom the group consisting of NB2/a, MN-1, and PC12 cells.
 4. The methodof claim 2 wherein the labeled GDNF is ¹²⁵I-GDNF.
 5. A competitive assayfor identifying compounds which bind to isolated GDNF receptorscomprising a) incubating said compounds with isolated c-RET receptors inthe presence of an excess of labeled GDNF; b) measuring the amount oflabeled GDNF bound to said receptors; and c) comparing amount labeledGDNF bound to said receptors to that of controls not incubated with saidcompounds.
 6. The method of claim 5 wherein the receptors arepolypeptides which bind GDNF selected from the group consisting ofpolypeptides about 55 kD, 70 kD, 135 kD, 155 kD, and 300 kD molecularweight.
 7. The method of claim 6 wherein the polypeptide is about 155 kDmolecular weight.
 8. The method of claim 5 wherein the isolated receptoris c-RET.
 9. The method of claim 5 wherein the labeled GDNF is¹²⁵I-GDNF.
 10. A method for identifying compounds which are GDNFhomologs comprising a) incubating said compounds with cells whichexpress c-RET receptors; and b) determining whether said compoundeffects tyrosine phosphorylation.
 11. The method of claim 10 whereinsaid cells are selected from the group consisting of PC12, MN-1, andNB2/a.
 12. A method for identifying compounds which are GDNF homologscomprising a) incubating said compounds with cells which express c-RETreceptors; and b) determining whether said compounds effect an increasein c-fos mRNA levels.
 13. The method of claim 12 wherein said cells areselected from the group consisting of PC12, MN-1, and NB2/a.
 14. Amethod for identifying compounds which are GDNF homologs comprising a)incubating said compounds with cells which express c-RET receptors undernon-permissive conditions for said cells; and b) determining the numberof surviving cells as compared to controls not incubated with saidcompounds.
 15. The method of claim 14 wherein said cells are selectedfrom the group consisting of PC12, MN-1, and NB2/a.
 16. A method foridentifying compounds which are GDNF analogs comprising a) incubatingsaid compounds with cells which express c-RET receptors in the presenceof concentrations of GDNF effective for phosphorylating tyrosine; and b)determining whether said compounds effect a decrease in the tyrosinephosphorylation as compared with controls not incubated with saidcompounds.
 17. The method of claim 16 wherein said cells are selectedfrom the group consisting of PC12, MN-1, and NB2/a.
 18. A method foridentifying compounds which are GDNF analogs comprising a) incubatingsaid compounds with cells which express c-RET receptors in the presenceof concentrations of GDNF effective for increasing c-fos mRNA levels;and b) determining whether said compounds effect a decrease in c-fosmRNA levels as compared with controls not incubated with said compounds.19. The method of claim 18 wherein said cells are selected from thegroup consisting of PC12, MN-1, and NB2/a.
 20. A method for identifyingcompounds which are GDNF analogs comprising a) incubating said compoundswith cells which express c-RET receptors under non-permissive conditionsfor said cells in the presence of amount of GDNF effective for cellsurvival; and b) determining the number of surviving cells as comparedwith controls not incubated with said compounds.
 21. The method of claim20 wherein said cells are selected from the group consisting of PC12,MN-1, and NB2/a.
 22. Isolated GDNFR-β comprising the amino acid sequenceof SEQ ID NO:2.
 23. Isolated GDNFR-β comprising the amino acid sequenceof SEQ ID NO:9.
 24. A compound comprising the amino acid sequence of SEQID NO:2.
 25. A compound comprising the amino acid sequence of SEQ IDNO:9.
 26. An ioslated nucleic acid having the sequence of SEQ ID No:5.27. An ioslated nucleic acid having the sequence of SEQ ID No: 10.